# Patent

## Title
Perfusion bioreactor for culturing CD200—placenta adherent cells

## Abstract

A method of culturing adherent cells from a placenta or adipose tissue is disclosed. The method comprises culturing the adherent cells from the placenta or adipose tissue under 3 dimensional (3D) culturing conditions which allow cell expansion, the conditions comprising perfusion.

## Background
RELATED APPLICATIONS
This application is a divisional application of U.S. application Ser. No. 13/061,538, filed Jul. 8, 2011, now U.S. Pat. No. 8,524,496, which is a national stage under 35 U.S.C. §371 of international application PCT/IL2009/00846, filed Sep. 1, 2009, which claims the benefit of U.S. Provisional Patent Application No. 61/202,050 filed Jan. 23, 2009 and U.S. Provisional Patent Application No. 61/136,375 filed Sep. 2, 2008.
The contents of all of the above documents are incorporated by reference as if fully set forth herein.


FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to adherent cells of placenta tissue and, more particularly, but not exclusively, to methods of culturing same and using same for treatment.
In recent years, considerable activity has focused on the therapeutic potential of mesenchymal stromal cells (MSCs) for various medical applications including tissue repair of damaged organs such as the brain, heart, bone and liver and in support of bone marrow transplantations (BMT). MSCs, a heterogeneous population of cells obtained from e.g. bone marrow, adipose tissue, placenta, and blood, is capable of differentiating into different types of cells (e.g. reticular endothelial cells, fibroblasts, adipocytes, osteogenic precursor cells) depending upon influences from various bioactive factors. Accordingly, MSCs have been widely studied in regenerative medicine as the foundation to build new tissues such as bone, cartilage and fat for the repair of injury or replacement of pathologic tissues and as treatment for genetic and acquired diseases [Fibbe and Noort, Ann N Y Acad Sci (2003) 996: 235-44; Horwitz et al., Cytotherapy (2005) 7(5): 393-5; Zimmet and Hare, Basic Res Cardiol (2005) 100(6): 471-81]. Furthermore, the multipotent ability of MSCs, their easy isolation and culture, as well as their high ex vivo expansion potential make them an attractive therapeutic tool [Fibbe and Noort, supra; Minguell et al. Exp Biol Med (Maywood) (2001) 226(6): 507-20].
An emerging body of data indicates that MSCs escape recognition of alloreactive cells and are considered to be immune privileged [Le Blanc et al., Exp Hematol (2003) 31(10): 890-6]. Having low immunogenicity, MSCs are not rejected by the patient's immune system and therefore are considered not to require HLA matching. Placental derived MSCs exhibit many markers common to MSCs isolated from other tissues, e.g. CD105, CD73, CD90 and CD29, and the lack of expression of hematopoietic, endothelial and trophoblastic-specific cell markers. Adipogenic, osteogenic, and neurogenic differentiation have been achieved after culturing placental derived MSCs under appropriate conditions [Yen et al., Stem Cells (2005) 23(1): 3-9]. Furthermore, MSCs isolated from placenta and cultured in vitro have been demonstrated to be immune privileged in a similar fashion as MSCs [Li et al., Cell Res (2005) 15(7): 539-47]. Thus, the placenta provides an ethically non-controversial and easily accessible source of MSCs for experimental and clinical applications [Zhang et al., Exp Hematol (2004) 32(7): 657-64]. In addition, the present inventors have previously devised three dimensional (3D) culturing conditions suitable for expansion of placental derived MSCs (WO/2007/108003).
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of culturing adherent cells from a placenta or adipose tissue, the method comprising culturing the adherent cells from the placenta or adipose tissue under 3 dimensional (3D) culturing conditions which allow cell expansion, the conditions comprising perfusion.
According to an aspect of some embodiments of the present invention there is provided a population of cells generated according to the above method.
According to an aspect of some embodiments of the present invention there is provided a population of cells comprising a gene expression profile essentially as described herein.
According to an aspect of some embodiments of the present invention there is provided a use of the population of cells, for the manufacture of a medicament identified for treating a condition which can benefit from cell or organ transplantation.
According to an aspect of some embodiments of the present invention there is provided a method of inducing tolerance and/or immunosuppression in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the adherent cells, thereby inducing tolerance and/or immunosuppression in the subject.
According to some embodiments of the invention, the perfusion is adjusted according to the glucose concentration of the culture medium.
According to some embodiments of the invention, the culture medium is maintained at a glucose concentration of about 550 mg/L.
According to some embodiments of the invention, the 3D culturing conditions comprise a 3D bioreactor.
According to some embodiments of the invention, the 3D culturing conditions comprise an adherent material selected from the group consisting of glass and plastic; polyester, polypropylene, polystyrene, dextran and collagen.
According to some embodiments of the invention, the 3D culturing conditions are effected for at least 3 days.
According to some embodiments of the invention, culturing of the cells is effected until at least 10% of the cells are proliferating.
According to some embodiments of the invention, at least 10% of the adherent cells are at a proliferative phase.
According to some embodiments of the invention, the adherent cells are capable of suppressing an immune reaction.
According to some embodiments of the invention, the adherent cells comprise a positive marker expression selected from the group consisting of CD73, CD90, CD29, CD105 and D7-fib.
According to some embodiments of the invention, the adherent cells comprise a negative marker expression selected from the group consisting of CD11b, CD34, HLA-DR, CD14, CD19, CD45, CD31, CD200 and KDR.
According to some embodiments of the invention, the adherent cells comprise a gene expression profile essentially as described herein.
According to some embodiments of the invention, the adherent cells are less committed to an osteogenic lineage as compared to adherent cells from bone marrow grown and allowed to differentiate under the same conditions.
According to some embodiments of the invention, the adherent cells are less committed to an adipogenic lineage as compared to adherent cells from bone marrow grown and allowed to differentiate under the same conditions.
According to some embodiments of the invention, the condition is selected from the group consisting of ischemia, peripheral arterial disease (PAD), critical limb ischemia (CLI), lower extremity ischemia, ischemic vascular disease, vascular disease of the kidney, ischemic heart disease, myocardial ischemia, coronary artery disease (CAD), atherosclerotic cardiovascular disease, left main coronary artery disease, arterial occlusive disease, peripheral ischemia, peripheral vascular disease, arteriosclerosis, ischemic brain disease, stroke, cerebral ischemia, cerebro vascular disease, retinopathy, retinal repair, remodeling disorder, von Hippel-Lindau syndrome, hereditary hemorrhagic telengiectasiaischemic vascular disease, Buerger's disease, ischemic renal disease, ischemic placenta, reproduction associated disorders, graft-versus-host disease, solid organ transplantation, hematopoietic stem cell transplantation, diabetes, connective tissue damage, cancer, pre-cancer, bone cancer, osteosarcoma, bone metastases, bone fracture, burn wound, articular cartilage defect, wound healing, deep wound, delayed wound-healing, delayed ulcer healing, subchondral-bone cyst, osteoporosis, osteoarthritis, degenerated bone, cartilage damage, articular cartilage defect, injured tendons, autoimmune disease, metabolic disorders, psoriasis, neuropathic pain, peripheral nerve injury, support of kidney transplantation and inflammatory disease.
According to some embodiments of the invention, the condition is selected from the group consisting of inflammatory bowel disease (IBD) and Crohn's disease.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIG. 1 is a flow chart depicting production of 3D adherent cell from placentas according to the present teachings (designated PLX-C cells).
FIG. 2 is a diagram of an exemplary bioreactor vessel and ports adapted from The New Brunswick Scientific web site.
FIGS. 3A-B depict cell cycle analysis of 3D adherent cells manufacture by Plurix (designated PLX, FIG. 3B) and by the present teachings (PLX-C, FIG. 3A). Cells were fixed in 70% EtOH O.N, centrifuged and re-suspended in a Propidium Iodide (PI) solution and then analyzed by FACS.
FIGS. 4A-D depict expression of fibroblast-typical markers but not expression of endothelial typical markers on PLX-C. FIG. 4A depicts negative expression of the endothelial marker CD31; FIG. 4B depicts negative expression of the endothelial marker KDR; FIG. 4C depicts positive expression of the human fibroblast marker (D7-FIB), wherein the red histograms for Isotype IgG1 (FITC) represent the negative control while the blue histograms represent the positively stained cells); and FIG. 4D depicts negative expression of the human CD200 marker (the pink histograms for Isotype IgG1 (PE) represent the negative control while the green histograms represent the positively stained cells).
FIGS. 5A-D depict expression of stimulatory and co-stimulatory molecules on PLX-C cells. FIG. 5A depicts PLX-C expression of CD80; FIG. 5B depicts PLX-C expression of CD86; FIG. 5C depicts PLX-C expression of CD40; and FIG. 5D depicts PLX-C expression of HLA-AB/C. Negative controls were prepared with relevant isotype fluorescence molecules. Of note, red histograms indicate PLX-C marker-expressing population of cells, blue histograms indicate bone marrow (BM) marker-expressing population of cells, and green histograms indicate mononuclear cell (MNC) marker expressing population of cells.
FIGS. 6A-B depict inhibition of lymphocyte proliferation by PLX-C. FIG. 6A depicts Mixed Lymphocyte Reaction (MLR) tests performed with 2×105 peripheral blood (PB) derived mononuclear cells (MNC, donor A) stimulated with equal amount of irradiated (3000 Rad) PB derived MNCs (donor B) followed by addition of increasing amounts of PLX-C cells to the cultures. Three replicates of each group were seeded in 96-well plates. Proliferation rate was measured by [3H]thymidine incorporation; FIG. 6B depict peripheral blood (PB) derived MNCs stimulated with ConA (1.5 mg /ml). Increasing amounts of PLX-C cells were added to the cultures. Three replicates of each group were seeded in 96-well plates. Proliferation rate was mesured by [3H] thymidine incorporation.
FIGS. 7A-C depict PLX-C regulation of pro-inflammatory and anti-inflammatory cytokine secretion following co-culture with peripheral blood cells. FIGS. 7A-B depict secretion of IFNγ (FIG. 7A) and TNFα (FIG. 7B) following co-culture of human derived MNCs (isolated from peripheral blood) stimulated with ConA with PLX-C; FIG. 7C depicts secretion of IFNγ, TNFα and IL-10 following co-culture of human derived MNCs (isolated from peripheral blood) stimulated with LPS with PLX-C. Supernatants were collected and subjected to cytokines analysis using ELISA.
FIGS. 8A-F are photographs depicting growth of bone marrow and placenta cells under osteogenesis or adipogenesis differentiation conditions. Bone marrow derived cells (FIGS. 8A-C) or placenta derived cells (FIGS. 8D-F) were plated in growth medium (FIGS. 8A and 8D), osteogenesis differentiation medium (FIGS. 8B and 8E) or adipogenesis differentiation medium (FIGS. 8C and 8F) in a 24 well plate coated with vitronectin and collagen. Medium was replaced every 3-4 days. At the end of growth period cells were fixed, stained and pictured as described in detail the Examples section which follows.
FIGS. 9A-F are photographs depicting growth of bone marrow and placenta cells under modified osteogenesis or adipogenesis differentiation conditions. Bone marrow derived cells (FIGS. 9A-C) or placenta derived cells (FIGS. 9D-F) were plated in growth medium (FIGS. 9A and 9D), osteogenesis differentiation medium (FIGS. 9B and 9E) or adipogenesis differentiation medium (FIGS. 9C and 9F) in a 24 well plate coated with vitronectin and collagen. Medium was replaced every 3-4 days. At the end of growth period cells were fixed, stained and pictured as described in detail the Examples section which follows.
FIG. 10 depicts the Luciferase expression vector used to infect PLX-C cells. Expression vector Lv33 from OmicsLink was used herein. The Luciferase gene was cloned into the ORF.
FIG. 11 depicts high Luciferase expression by infected PLX-C cells. Cells were infected with the Luciferase expression vector and visualized by the IVIS system 48 hours post infection. Of note, cells exhibited high levels of Luciferase expression.
FIGS. 12A-D depict injection of 2×106 Luciferase expressing PLX-C cells into SCID/Beige mice. One mouse was injected IM and one IV. The injected mice were monitored using the IVIS system in order to asses the in vivo biodistribution of PLX-C. IVIS results of days 1 (FIG. 12A), day 4 (FIG. 12B), day 6 (FIG. 12C) and day 22 (FIG. 12D) are presented.



DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to adherent cells of placenta tissue and, more particularly, but not exclusively, to methods of culturing same and using same for treatment.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
While reducing the present invention to practice, the present inventors have uncovered that culturing placenta derived adherent cells under three dimensional (3D) culturing conditions, comprising perfusion, generate large amounts of adherent cells which are characterized by a distinctive gene expression profile, are capable of suppressing an immune response and are highly proliferative. Thus, these placenta adherent cells may be used for therapeutic applications.
As is illustrated herein below and in Example 1-8 of the Examples section which follows, the present inventors were able to expand placenta—derived adherent cells under 3D conditions. The 3D conditions of the present invention comprise perfusion of the cell medium within the bioreactor (see Example 2). As is shown in Example 3, the placenta adherent cells of the present invention comprise stromal stem cell properties, e.g. they express cellular markers typical of stromal stem cells, and comprise immunosuppressive properties. Furthermore, these cells are highly proliferative (28% of cells were in S and G2/M phases) and are retained in the body for a few weeks after administration (see Examples 3 and 8), suggesting these cells may be used for treatment.
In addition, in their 2D stage, the placenta derived adherent cells of the present invention did not differentiate into osteocytes (Examples 4-5) or adipocytes (Examples 6-7), in sharp contrast to bone marrow adherent cells of which a high percentage (over 50%) underwent differentiation when grown under the same conditions.
Thus, according to one aspect of the present invention there is provided a method of culturing adherent cells from a placenta or adipose tissue, the method comprising culturing the adherent cells from the placenta or adipose tissue under three dimensional (3D) culturing conditions which allow cell expansion, the conditions comprising perfusion.
As used herein the phrase “adherent cells” refers to a homogeneous or heterogeneous population of cells which are anchorage dependent, i.e., require attachment to a surface in order to grow in vitro.
As used herein the phrase “adipose tissue” refers to a connective tissue which comprises fat cells (adipocytes).
As used herein the term “placenta tissue” refers to any portion of the mammalian organ which lines the uterine wall and during pregnancy envelopes the fetus, to which it is attached by the umbilical cord. Following birth, the placenta is expelled (and is referred to as a post partum placenta). In an exemplary embodiment, placenta refers to whole placenta.
According to the present teachings, placenta or adipose tissue derived adherent cells are propagated using three dimensional (3D) culturing conditions.
As used herein the phrase “three dimensional culture” refers to a culture in which the cells are disposed to conditions which are compatible with cell growth including a scaffold which allows cell to cell contacts in three dimensions. It is well appreciated that the in situ environment of a cell in a living organism (or a tissue) is in a three dimensional architecture. Cells are surrounded by other cells. They are held in a complex network of extra cellular matrix nanoscale fibers that allows the establishment of various local microenvironments. Their extra cellular ligands mediate not only the attachment to the basal membrane but also access to a variety of vascular and lymphatic vessels. Oxygen, hormones and nutrients are ferried to cells and waste products are carried away. The conditions in the three dimensional culture of the invention are designed to mimic such an environment as is further exemplified below.
It will be appreciated that the conditions of the three-dimensional culture are such that enable expansion of the adherent cells.
As used herein the terms “expanding” and “expansion” refer to substantially differentiation-less maintenance of the cells and ultimately cell growth, i.e., increase of a cell population (e.g., at least 2 fold) without differentiation accompanying such increase.
As used herein the terms “maintaining” and “maintenance” refer to substantially differentiation-less cell renewal, i.e., substantially stationary cell population without differentiation accompanying such stationarity.
As mentioned, the adherent cells of this aspect of the invention are retrieved from a placental or adipose tissue.
Placental cells may be obtained from a full-term or pre-term placenta. Placenta is preferably collected once it has been ex blooded. The placenta is preferably perfused for a period of time sufficient to remove residual cells (e.g., blood).
The term “perfuse” or “perfusion” used herein refers to the act of pouring or passaging a fluid over the placenta or in the later stages of the method over the cultured cells. The placental tissue may be from any mammal; for example, the placental tissue is human. A convenient source of placental tissue is from a post partum placenta (e.g., 1-6 hours), however, the source of placental tissue or cells or the method of isolation of placental tissue is not critical to the invention.
Placenta derived adherent cells may be obtained from both fetal (i.e., amnion, chorion, chorionic villi or inner parts of the placenta, see Example 1) and maternal (i.e., decidua basalis, and decidua parietalis) parts of the placenta. Tissue specimens are washed in a physiological buffer [e.g., phosphate-buffered saline (PBS) or Hank's buffer]. Single-cell suspensions are made by treating the tissue with a digestive enzyme (see below) or/and mincing and flushing the tissue parts through a nylon filter or by gentle pipetting (Falcon, Becton, Dickinson, San Jose, Calif.) with washing medium.
It will be appreciated that adherent cells may be derived from adipose tissue. Adipose tissue derived adherent cells may be isolated by a variety of methods known to those skilled in the art. For example, such methods are described in U.S. Pat. No. 6,153,432. The adipose tissue may be derived from omental/visceral, mammary, gonadal, or other adipose tissue sites. One source of adipose tissue is omental adipose. In humans, the adipose is typically isolated by liposuction.
Isolated adherent cells from placenta or adipose tissue may be derived by treating the tissue with a digestive enzyme such as collagenase, trypsin and/or dispase; and/or effective concentrations of hyaluronidase or DNAse; and ethylenediaminetetra-acetic acid (EDTA); at temperatures between 25-50° C., for periods of between 10 minutes to 3 hours. The cells may then be passed through a nylon or cheesecloth mesh filter of between 20 microns to 1 mm. Cells are centrifuged at speeds of between 100 to 3000×g for periods of between 1 minutes to 1 hour at temperatures of between 4-50° C. (see U.S. Pat. No. 7,078,230).
Cell retrieval from placenta or adipose tissue is preferably effected under aseptic conditions. Once isolated cells are obtained, they are allowed to adhere to an adherent material (e.g., configured as a surface) to thereby isolate adherent cells.
As used herein “an adherent material” refers to a synthetic, naturally occurring or a combination of same of a non-cytotoxic (i.e., biologically compatible) material having a chemical structure (e.g., charged surface exposed groups) which may retain the cells on a surface.
Examples of adherent materials which may be used in accordance with this aspect of the invention include, but are not limited to, a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, a matrigel, an extra cellular matrix component (e.g., fibronectin, vitronectin, chondronectin, laminin), a collagen, a poly L lactic acid, a dextran and an inert metal fiber.
Further steps of purification or enrichment for stromal stem cells may be effected using methods which are well known in the art (such as by FACS using stromal stem cell marker expression, as further described herein below).
Non-limiting examples of base media useful in culturing according to the invention include Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), F10(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E-with Earle's sale base), Medium M199 (M199H-with Hank's salt base), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and Minimum Essential Medium Eagle (MEM-NAA with non essential amino acids), among numerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. A preferred medium for use in the invention is DMEM. These and other useful media are available from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet HaEmek, Israel, among others. A number of these media are summarized in Methods in Enzymology, Volume LVIII, “Cell Culture”, pp. 62 72, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.
The medium may be supplemented such as with serum such as fetal serum of bovine or human or other species, and optionally or alternatively, growth factors, vitamins (e.g. ascorbic acid), cytokines, salts (e.g. B-glycerophosphate), steroids (e.g. dexamethasone) and hormones e.g., growth hormone, erythropoeitin, thrombopoietin, interleukin 3, interleukin 6, interleukin 7, macrophage colony stimulating factor, c-kit ligand/stem cell factor, osteoprotegerin ligand, insulin, insulin like growth factors, epidermal growth factor, fibroblast growth factor, nerve growth factor, cilary neurotrophic factor, platelet derived growth factor, and bone morphogenetic protein at concentrations of between picogram/ml to milligram/ml levels.
It is further recognized that additional components may be added to the culture medium. Such components may be antibiotics, antimycotics, albumin, amino acids, and other components known to the art for the culture of cells. Additionally, components may be added to enhance the differentiation process when needed (see further below).
As mentioned, once adherent cells are at hand they may be passaged to two dimensional or three dimensional settings (see Examples 1 and 2 of the Examples section which follows). It will be appreciated though, that the cells may be transferred to a 3D-configured matrix immediately after isolation or alternatively, may be passaged to three dimensional settings following two dimensional (2D) conditions (as mentioned hereinabove).
It will be appreciated that during the 2D culturing conditions, the adherent cells may be continuously passaged. According to an embodiment of the present invention, the cells may be passaged for at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages or at least 8 passages. It will be appreciated that cells are typically passaged when the culture reaches about 70-90% confluence, typically after 3-7 days (e.g., 3-5 days, 1-3 doublings). Moreover, under the 2D culturing conditions, the cells may be grown in a culture medium devoid of antibiotic supplements from at least passage 2, at least passage 3, or at least passage 4.
Thus, the adherent material of this aspect of the invention is configured for 3D culturing thereby providing a growth matrix that substantially increases the available attachment surface for the adherence of the cells so as to mimic the infrastructure of the tissue (e.g., placenta).
For high scale production, culturing can be effected in a 3D bioreactor.
Examples of such bioreactors include, but are not limited to, a plug flow bioreactor, a continuous stirred tank bioreactor, a stationary-bed bioreactor (packed bed bioreactor) and a fluidized bed bioreactor.
As shown in Example 2 of the Examples section, the Celligen bioreactor is capable of 3D expansion of adherent cells under controlled conditions (e.g. pH, temperature and oxygen levels) and with constant cell growth medium perfusion. Furthermore, the cell cultures can be monitored for concentration levels of glucose, lactate, glutamine, glutamate and ammonium. The glucose consumption rate and the lactate formation rate of the adherent cells enable to measure cell growth rate and to determine the harvest time.
Other 3D bioreactors that can be used with the invention include, but are not limited to, a continuous stirred tank bioreactor, where a culture medium is continuously fed into the bioreactor and the used medium is continuously drawn out, to maintain a time-constant steady state within the bioreactor. The stirred tank bioreactor may be used with fluidized bed (suspended carriers) or a fibrous bed basket (which is available for example at New Brunswick Scientific Co., Edison, N.J.), a stationary-bed bioreactor, an air-lift bioreactor, where air is typically fed into the bottom of a central draught tube flowing up while forming bubbles, and disengaging exhaust gas at the top of the column, a bioreactor with Polyactive foams [as described in Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)], a porous scaffolds in a Radial-flow perfusion bioreactor [as described in Kitagawa et al., Biotechnology and Bioengineering 93(5): 947-954 (2006)], a radial flow bioreactor with scaffold or carriers, a hollow fiber bioreactor, and micro carriers. Other bioreactors which can be used in accordance with the invention are described in U.S. Pat. Nos. 6,277,151, 6,197,575, 6,139,578, 6,132,463, 5,902,741 and 5,629,186.
In an exemplary embodiment a total of 150±50×106 cells are seeded, 3-7×106 cell/gr carrier are seeded, or 0.06-0.13×106 cell/ml are seeded. According to an exemplary embodiment, cell seeding is effected at 1400-7000 cells/cm2 FibraCel disks.
Cells can be harvested when at least about 10% of cells are proliferating while avoiding uncontrolled differentiation and senescence.
Culturing is effected for at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 20 days, a month or even more. It will be appreciated that culturing in a bioreactor may prolong this period. Culturing of the adherent cells in the 3D culture can be effected under a continuous flow of a culture medium. Passaging may also be effected to increase cell number. It will be appreciated that culture medium may be changed in order to prolong and improve culturing conditions.
According to an embodiment of the present invention, the cell culturing is effected under perfusion of the culture medium. Typically, the perfusion rate is determined by the glucose concentration in the culture medium of the adherent cells. Thus, according to the present teachings, the culture medium may be changed when the glucose concentration is about 500 mg/L, about 550 mg/L, or about 600 mg/L.
Adherent cells of some embodiments of the present invention comprise at least about 10%, 28%, 30%, 50%, 80% or more proliferative cells (as can be assayed by FACS monitoring S and G2/M phases).
Adherent cells of some embodiments of the invention may comprise at least one “stromal stem cell phenotype”.
As used herein “a stromal stem cell phenotype” refers to a structural or functional phenotype typical of a bone-marrow derived stromal (i.e., mesenchymal) stem cell.
As used herein the phrase “stem cell” refers to a cell which is not terminally differentiated.
Thus for example, the cells may have a spindle shape. Alternatively or additionally the cells may express a marker or a collection of markers (e.g. surface marker) typical to stromal stem cells. Examples of stromal stem cell surface markers (positive and negative) include but are not limited to CD105+, CD29+, CD44+, CD73+, CD90+, D7-fib+, CD3−, CD4−, CD34−, CD45−, CD80−, CD5-, CD20-, CD11B−, CD14−, CD19−, CD79−, HLA-DR−, CD31−, KDR−, and FMC7−. Other stromal stem cell markers include but are not limited to tyrosine hydroxylase, nestin and H-NF.
Adherent cells of placenta tissue generated according to the present teachings have a gene expression profile essentially as described in Example 3 of the Examples section which follows.
Examples of functional phenotypes typical of stromal stem cells include, but are not limited to, T cell suppression activity (they don't stimulate T cells and conversely suppress same) and hematopoietic stem cell support activity.
According to an exemplary embodiment, the adherent cells of the present invention are less committed to differentiation into osteogenic or adipogenic lineages as compared to adherent cells from the bone marrow grown and differentiated under the same conditions (see Examples 4-5 and Examples 6-7, respectively).
As is shown in Examples 3 of the Examples section which follows, the adherent cells of the present invention were found to suppress the immune reaction of human mononuclear cells in a mixed lymphocyte reaction (MLR) assay, thus exhibit biological activities which may be preferentially used in the clinic (e.g., T cell suppression activity, hematopoietic stem cell support activity).
According to one embodiment of the invention, the adherent cells of the invention are capable of suppressing immune reaction in a subject.
As used herein the phrase “suppressing immune reaction in a subject” refers to decreasing or inhibiting the immune reaction occurring in a subject in response to an antigen (e.g., a foreign cell or a portion thereof). The immune response which can be suppressed by the adherent cells include the humoral immune responses, and cellular immune responses, which involve specific recognition of pathogen antigens via antibodies and T-lymphocytes (proliferation of T cells), respectively.
The populations of cells generated according to the present teachings may be used for treating a condition which can benefit from cell or organ transplantation.
As used herein, the term “condition” refers to any pathology (disease, condition, syndrome or disorder) which may benefit from cell (e.g. stem cell) or organ transplantation. Examples include ischemic conditions, cardiovascular conditions, nervous system conditions, gastrointestinal tract conditions, orthopedic conditions, hematopoietic conditions, renal conditions and hepatic conditions, such as but are not limited to, peripheral arterial disease (PAD), such as limb ischemia and critical limb ischemia (CLI), lower extremity ischemia, ischemic vascular disease, ischemic heart disease, myocardial ischemia, acute myocardial infarction (MI), coronary artery disease (CAD), atherosclerotic cardiovascular disease, left main coronary artery disease, arterial occlusive disease, peripheral ischemia, peripheral vascular disease, arteriosclerosis, ischemic brain disease, stroke, cerebral ischemia, cerebro vascular disease, retinopathy, retinal repair, remodeling disorder, von Hippel-Lindau syndrome, hereditary hemorrhagic telengiectasiaischemic vascular disease, Buerger's disease, diabetes, vascular disease of the kidney, ischemic renal disease, liver disease, ischemic placenta, reproduction associated disorders, graft-versus-host disease (GVHD), solid organ transplant, hematopoietic stem cell transplantation (HSCT), metabolic disorders, inflammatory conditions of the gastrointestinal (GI) tract [e.g. inflammatory bowel disease (IBD)], ulcerative colitis, delayed wound-healing, delayed ulcer healing, cancer (e.g. breast cancer), pre-cancer, conditions characterized by connective tissue damage such as bone cancer, osteosarcoma, bone metastases, bone fracture, degenerative disc disease, osteogenesis imperfecta (OI), burn, burn wound, articular cartilage defect, wound healing, deep wound, delayed wound-healing, delayed ulcer healing, subchondral-bone cyst, osteoporosis, osteoarthritis (OA), degenerated bone, cartilage damage, articular cartilage defect, injured tendons (e.g. overstrain-induced injuries of tendons) and injured ligaments.
It will be appreciated that the adherent cells of the present invention are capable of inducing immunosuppression and/or tolerance in a subject. Thus, the adherent cells may be used to treat any condition in need of immunosuppression and/or tolerance. Such conditions included, but are not limited to, autoimmune diseases and inflammatory diseases (including acute and chronic inflammatory diseases) including, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases (e.g., neuropathic pain, peripheral nerve injury), muscular diseases, nephric diseases, support for renal transplantation, diseases related to reproduction, connective tissue diseases and systemic diseases.
Examples of autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug. 25; 112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000; 26 (2):157), necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel L H. Ann Med Interne (Paris). 2000 May; 151 (3):178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4):171), antibody-induced heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun. 17; 83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 April-June; 14 (2):114; Semple J W. et al., Blood 1996 May 15; 87 (10):4245), autoimmune hemolytic anemia (Efremov D G. et al., Leuk Lymphoma 1998 Jan.; 28 (3-4):285; Sallah S. et al., Ann Hematol 1997 March; 74 (3):139), cardiac autoimmunity in Chagas' disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct. 15; 98 (8):1709) and anti-helper T lymphocyte autoimmunity (Caporossi A P. et al., Viral Immunol 1998; 11 (1):9).
Examples of autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 July; 15 (3):791; Tisch R, McDevitt H O. Proc Natl Acad Sci units S A 1994 Jan. 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189).
Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome. diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth G S. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 October; 34 Suppl:S125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 June; 29 (2):339; Sakata S. et al., Mol Cell Endocrinol 1993 March; 92 (1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec. 15; 165 (12):7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 August; 57 (8):1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 August; 57 (8):1759), ovarian autoimmunity (Garza K M. et al., J Reprod Immunol 1998 February; 37 (2):87), autoimmune anti-sperm infertility (Diekman A B. et al., Am J Reprod Immunol. 2000 Mar; 43 (3):134), autoimmune prostatitis (Alexander R B. et al., Urology 1997 December; 50 (6):893) and Type I autoimmune polyglandular syndrome (Hara T. et al., Blood. 1991 Mar. 1; 77 (5):1127).
Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 January; 23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122), colitis, ileitis and Crohn's disease.
Examples of autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid, psoriasis and pemphigus foliaceus.
Examples of autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 March; 54 (3):382), primary biliary cirrhosis (Jones D E. Clin Sci (Colch) 1996 November; 91 (5):551; Strassburg C P. et al., Eur J Gastroenterol Hepatol. 1999 June; 11 (6):595) and autoimmune hepatitis (Manns M P. J Hepatol 2000 August; 33 (2): 326).
Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross A H. et al., J Neuroimmunol 2001 Jan. 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (Infante A J. And Kraig E, Int Rev Immunol 1999; 18 (1-2):83; Oshima M. et al., Eur J Immunol 1990 December; 20 (12):2563), neuropathies, motor neuropathies (Kornberg A J. J Clin Neurosci. 2000 May; 7 (3):191); Guillain-Barre syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 April; 319 (4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 April; 319 (4):204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra H S. et al., Proc Natl Acad Sci units S A 2001 Mar. 27; 98 (7):3988); non-paraneoplastic stiff man syndrome, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome and autoimmune polyendocrinopathies (Antoine J C. and Honnorat J. Rev Neurol (Paris) 2000 January; 156 (1):23); dysimmune neuropathies (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999; 50:419); acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13; 841:482), neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May; 57 (5):544) and neurodegenerative diseases.
Examples of autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 September; 123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 June; 53 (5-6):234).
Examples of autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly C J. J Am Soc Nephrol 1990 August; 1 (2):140).
Examples of autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al., Lupus 1998; 7 Suppl 2:S107-9).
Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo T J. et al., Cell Immunol 1994 August; 157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec. 29; 830:266).
Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 March; 6 (2):156); Chan O T. et al., Immunol Rev 1999 June; 169:107).
Furthermore, the adherent cells of the present invention may be used to treat diseases associated with transplantation of a graft including, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.
As used herein the term “treating” refers to inhibiting or arresting the development of a pathology and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology. The term “treating” may also refer to alleviating or diminishing a symptom associated with the pathology.
The subject treated by the adherent cells may be any subject (e.g., a mammal), such as a human subject or a domesticated animal including, but not limited to, horses (i.e. equine), cattle, goat, sheep, pig, dog, cat, camel, alpaca, llama and yak who is diagnosed with or suffers from the pathology and can benefit from stromal stem cell transplantation.
Methods of deriving lineage specific cells from the adherent cells (e.g. stromal stem cells) of the invention are well known in the art. See for example, U.S. Pat. Nos. 5,486,359, 5,942,225, 5,736,396, 5,908,784 and 5,902,741.
The adherent cells may be naïve or may be genetically modified such as to derive a lineage of interest (see U.S. Pat. Appl. No. 20030219423).
The cells may be of autologous or non-autologous source. A non-autologous source may be allogeneic or xenogeneic. Cells may be used as fresh or frozen (e.g., cryo-preserved) preparations.
Depending on the medical condition, the subject may be administered with additional chemical drugs (e.g., immunomodulatory, chemotherapy etc.) or cells.
Even though the cells are characterized by immuno-suppressive activity, they may still provoke host or donor-derived undesirable immune response. Approaches have been developed to reduce the likelihood of rejection of non-autologous cells or GvHD. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.
Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.
For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56). Other microcapsules are based on alginate, a marine polysaccharide (Sambanis,
A. Encapsulated islets in diabetes treatment. Diabetes Technol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).
Examples of immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNFalpha blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.
Furthermore, it will be appreciated that the cells can be administered either per se or, preferably as a part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.
As used herein a “pharmaceutical composition” refers to a preparation of the adherent cells of the invention (i.e., adherent cells), with other chemical components such as pharmaceutically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the cells to a subject.
Hereinafter, the term “pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. Examples, without limitations, of carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water.
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
Pharmaceutical compositions for use in accordance with the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological salt buffer, or freezing medium containing cryopreservents.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations. However, the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions including the preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
Compositions of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
It is expected that during the life of a patent maturing from this application many relevant three dimensional cultures will be developed and the scope of the term three dimensional cultures is intended to include all such new technologies a priori.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Example 1
Production of Placenta Derived Adherent Cells by the Methods of WO/2007/108003
Adherent cells were produced as was previously described (see WO/2007/108003) in a bioreactor system containing 3D carriers to produce adherent cells (designated herein as PLX).
Materials and Experimental Procedures
Placenta derived adherent cells—Inner parts of a full-term delivery placenta (Bnei Zion medical center, Haifa, Israel) were cut under aseptic conditions, washed 3 times with Hank's Buffer and incubated for 3 hours at 37° C. with 0.1% Collagenase (1 mg/ml tissue; Sigma—Aldrich, St. Lewis, Mo.). Using gentle pipetting, suspended cells were then washed with DMEM supplemented with 10% FCS, Pen-Strep-Nystatin mixture (100 U/ml:100 μg/ml:1.25 un/ml) and 2 mM L-glutamine, seeded in 75 cm2 flasks and incubated at 37° C. in a tissue culture incubator under humidified condition with 5% CO2.
Two Dimensional (2D) Cell Growth
Cells were allowed to adhere to a plastic surface for 48-72 hours after which the media was changed every 3-4 days. After 2-3 passages, the cells were cryopreserved, thawed and seeded for a secondary growth in flasks. When reaching 60-80% confluence cells were detached from the growth flask using 0.25% trypsin-EDTA and seeded into new flasks (usually every 3-5 days), for another 2-5 passages. Cultured cells were thereafter collected for analysis or for culturing in bioreactors.
PluriXTM Plug Flow bioreactor—The PluriXTM Plug Flow bioreactor (Pluristem, Haifa, Israel; as was previously described in U.S. Pat. No. 6,911,201 and WO/2007/108003), was loaded with 1-100 ml packed 3D porrosive carriers (4 mm in diameter) made of a non woven fabric matrix of polyester. These carriers enable the propagation of large cell numbers in a relatively small volume. Glassware was designed and manufactured by Pluristem (Pluristem, Haifa, Israel). The bioreactor was maintained in an incubator of 37° C., with flow rate regulated and monitored by a valve and peristaltic pump. The bioreactor contains a sampling and injection point, allowing the sequential seeding of cells. Culture medium was supplied at pH 6.7-7.4 from a reservoir. The reservoir was supplied by a filtered gas mixture containing air/CO2/O2 at differing proportions, depending on cell density in the bioreactor. The O2 proportion was suited to the level of dissolved O2 at the bioreactor exit, determined by a monitor. The gas mixture was supplied to the reservoir via silicone tubes or diffuser (Degania Bet, Emek Hayarden, Israel). The culture medium was passed through a separating container which enables collection of circulating, non-adherent cells. Circulation of the medium was obtained by a peristaltic pump. The bioreactor was further equipped with an additional sampling point and containers for continuous medium exchange.
Production of PLX adherent cells—Non-confluent primary human adherent 2D cell cultures, grown as described above, were trypsinized, washed, resuspended in DMEM supplemented with 10% FBS, Pen-Strep-Nystatin mixture (100 U/M:100 ug/ml:1.25 un/ml) and 2 mM L-glutamine, and seeded (103-105 cells/ml) via an injection point onto the 3D carriers in a sterile Plug Flow bioreactor. Prior to inoculation, bioreactor was filled with PBS-Ca-Mg (Biological Industries, Beit Ha'emek, Israel), autoclaved (120° C., 30 min) and washed with Dulbecco's growth medium containing 10% heat-inactivated fetal calf serum and a Pen-Strep-Nystatin mixture (100 U/ml:100 ug/ml:1.25 un/ml). Flow was kept at a rate of 0.1-5 ml/min. Seeding process involved cease of circulation for 2-48 hrs, thereby allowing the cells to settle on the carriers. Bioreactor was kept under controlled temperature (37° C.) and pH conditions (pH=6.7-7.4); using an incubator supplied with sterile air and CO2 as needed. Growth medium was replaced 2-3 times a week. Circulation medium was replaced with fresh DMEM media, every 4 hr to 7 days. At a density of 1×106-1×107 cells/ml (following 12-40 days of growth), total medium volume was removed from the bioreactor and bioreactor and carriers were washed 3-5 times with PBS. PLX adherent cells were then detached from the carriers with Trypsin-EDTA; (Biological Industries, Beit Ha'emek, Israel; 3-15 minutes with gentle agitation, 1-5 times), and were thereafter resuspended in DMEM and cryopreserved.
Example 2
Production of the Placenta Derived Adherent Cells of the Present Invention
PLX-C adherent cells were produced by the present invention which exhibit different characteristics then the above described PLX adherent cells.
Materials and Experimental Methods
CelligenTM Plug Flow bioreactor—The production of adherent cells of the present invention by CelligenTM (PLX-C cells) is composed of several major steps as illustrated in FIG. 1. The process starts by collection of a placenta from a planned caesarean section at term.
Adherent cells are then isolated from whole placentas, grown in tissue culture flasks (2D cultures), harvested and stored in liquid nitrogen as 2D-Cell Stock (2DCS), the appropriate amount of 2DCS are thawed, washed and seeded onto carriers in bioreactors for further expansion as 3D-culture. After 4-12 days of growth in the bioreactors, cells are harvested and cryopreserved in gas phase of liquid nitrogen as PLX-C.
Receipt of Human Tissue
All placentas obtained were received from the maternity ward under approval of the Helsinki Committee of the medical facility. Accordingly, all placenta donors signed an informed consent and Donor Screening and Donor Testing was performed. Immediately after taking the placenta from the donor (during the caesarean procedure), it was placed in a sterile plastic bag and then in a temperature-preserving box with ice packs.
Recovery and Processing of Adherent Cells
To initiate the process, the placenta tissue was cut into pieces under aseptic conditions under laminar flow hood, washed with Hank's buffer solution and incubated for 2-5 hours at 37° C. with 0.1% Collagenase (1 mg Collagenase/ml tissue). 2D cell medium (2D-Medium comprising DMEM supplemented with 10% FBS, fungizone 0.25 μg/ml and Gentamycin 50 μg/ml) was added and the digested tissue was roughly filtered through a sterile metal strainer, collected in a sterile beaker and centrifuged (10 minutes, 1200 RPM, 4° C.). Using gentle pippeting, suspended cells were then diluted with 2D-Medium supplemented with antibiotics, seeded in 175 cm2 flasks and incubated at 37° C. in a tissue culture incubator under humidified condition supplemented with 5% CO2. Following 2-3 days, in which the cells were allowed to adhere to the flask surface, they were washed with PBS and 2D-Medium was added.
Two Dimensional (2D) Cell Growth
Prior to the first passage, growth medium samples of 10% of the total flask number in quarantine was pooled and taken for mycoplasma testing (IPC2). If cells were found to be negative for Mycoplasma (EZ-PCR Mycoplasma kit, Biological Industries, Israel), cells were released from quarantine. After 1-2 additional passages using 2D-Medium supplemented with antibiotics, cells were transferred to the 2D production clean room (2DP). Once in Room 2DP, culture was continued for another 3-6 passages using 2D-Medium without antibiotics. Throughout the process, cultures were grown in a tissue culture incubator under humidified conditions with 5% CO2 at 37° C. After a total of 6-9 passages (9-17 cell doublings), cells were collected and cryopreserved as the 2D-Cell Stock (2DCS).
The first passage was usually carried out after 7-15 days. Beginning at passage 2 and continuing until passage 6-9, cells were passaged when the culture reached 70-90% confluence, usually after 4-5 days (1.5-2 doublings). The cells were detached from the flasks using 0.25% trypsin-EDTA (4 minutes at 37° C.) and seeded in a culture density of 4±0.5×103 cells/cm2. The size of the tissue culture flasks raised as the passages proceed. The culturing process started in 175 cm2 tissue culture flask, continued in 500 cm2 (Triple flask) and finally the cells were seeded into Cell Factory 10 tray (6320 cm2).
Prior to cryopreservation, at the end of 2DCS growth period, the growth medium was collected and the sample was prepared to be sent to an approved GLP laboratory for Mycoplasma test (IPC 4).
Cryopreservation Procedure for 2D-Cell-Stock Product
For 2DCS cryopreservation, 2D-cultured cells were collected under aseptic conditions using 0.25% trypsin-EDTA. The cells were centrifuged (1200 RPM, 10′, 4° C.), counted and re-suspended in 2D-Medium.
For freezing, cell suspensions were diluted 1:1 with 2D-Freezing Mixture (final concentrations was 10% DMSO, 40% FBS and 50% 2D-Medium). Approximately 1.5-2.5×109 cells were manufactured from one placenta. 4 ml of the cells were stored at a final concentration of 10×106/ml in 5 ml cryopreservation polypropylene vials. The vials were labeled and transferred to a controlled rate freezer for a graduated temperature reducing process (1° C./min), after which they were transferred to storage in gas-phase of a liquid nitrogen freezer. This material was referred to as the 2D-Cell Stock (2DCS) batch.
Initiation of the Three Dimensional (3D) Culture Procedures
To begin 3D culture, an appropriate amount (150±50×106) of cells from 2DCS were thawed in the 2DP room and washed with 3D-Medium (DMEM with 10% FBS and 20 Mm Hepes) to remove DMSO prior to seeding in the prepared-in-advanced bioreactor systems. The content of each 2DCS vial was pipetted and diluted 1:9 with pre-warmed (37° C.) 3D-Medium. The cells were centrifuged (1200 RPM, 10′, 4° C.) and re-suspended again in 50-100 ml pre-warmed (37° C.) 3D-Medium in a 250 ml sterile bottle. A sample was taken and cells were counted using a Trypan Blue stain in order to determine cell number and viability. The cell suspension was transferred under a laminar flow hood into a 0.5 L seeding bottle. From the seeding bottle the cell suspension was transferred via sterile tubing to the bioreactor by gravitation.
Production of Adherent Cells in the Celligen Bioreactor (PLX-C)
Bioreactor Description
3D growth phase was performed using an automatic CelliGen Plus® or BIOFLO 310 bioreactor system [(New Brunswick Scientific (NBS)] depicted in FIG. 2. The bioreactor system was used for cultivation of cell culture, in which conditions were suitable for high cell concentrations. The cultivation process was carried out using a bioreactor in a perfusion mode. The lab scale bioreactor was constructed of two main systems—the control system and the bioreactor itself (vessel and accessories). The parameters of the process were monitored and controlled by a control console which included connectors for probes, motor and pumps, control loops for Dissolved Oxygen (DO), pH, perfusion and agitation (with a motor), a gases control system, water circulation and heating system for temperature control and an operator interface. The controlled process parameters (such as temperature, pH, DO etc.) could be displayed on the operator interface and monitored by a designated controller.
Cell Culture Growth Procedure in the Bioreactors
As noted in the section hereinabove, 150±50×106 cells from the cryopreserved 2DCS were thawed, washed and seeded in a sterile bioreactor. The bioreactor contained 30-50 gr carriers (FibraCel® disks, NBS), made of Polyester and Polypropylene and 1.5±0.1 L 3D-Medium. The growth medium in the bioreactor was kept at the following conditions: 37° C., 70% Dissolved Oxygen (DO) and pH 7.3. Filtered gases (Air, CO2. N2 and O2) were supplied as determined by the control system in order to keep the DO value at 70% and the pH value at 7.3. For the first 24 hours, the medium was agitated at 50 Rounds Per Minutes (RPM) and increased up to 200 RPM by day 2. For the first 2-3 days, the cells were grown in a batch mode. Perfusion was initiated when the medium glucose concentration decreased below 550 mg/liter. The medium was pumped from the feeding container to the bioreactor using sterile silicone tubing. All tubing connections were performed under laminar flow using sterile connectors. The perfusion was adjusted on a daily basis in order to keep the glucose concentration constant at approximately 550±50 mg\liter. A sample of the growth medium was taken every 1-2 days for glucose, lactate, glutamine, glutamate and ammonium concentration determination (BioProfile 400 analyzer, Nova Biomedical). The glucose consumption rate and the lactate formation rate of the cell culture enabled to measure cell growth rate. These parameters were used to determine the harvest time based on accumulated experimental data.
Harvest of the 3D Grown PLX-C Cells from the Bioreactor
The cell harvest process started at the end of the growth phase (4-12 days). Two samples of the growth medium were collected. One sample was prepared to be sent to an approved GLP laboratory for Mycoplasma testing according to USP and Eu standards. This medium sample was considered as part of the Mycoplasma testing of the final product and the results were considered as part of the criteria for product release.
The 3D-grown culture was harvested in the Class-100 laminar area in room 3DP as follows:
The bioreactor vessel was emptied using gravitation via tubing to a waste container. The bioreactor vessel was then refilled with 1.5 L pre-warmed PBS (37° C.). The agitation speed was increased to 150 RPM for 2 minutes. The PBS was drained via tubing by pressure or gravity to the waste bottle. The washing procedure was repeated twice.
In order to release the cells from the carriers, 1.5 L pre-warmed to 37° C. Trypsin-EDTA (Trypsin 0.25%, EDTA 1 mM) was added to the bioreactor vessel and carriers were agitated for 1-4 minutes in 150 RPM, 37° C. 250 ml FBS was added to the bioreactor vessel and the cell suspension was collected to a 5 L sterile container. Cell suspension was divided to 500 ml sterile centrifuge tubes which were centrifuged (1200 RPM, 10′, 4° C.) and re-suspended in cryopreservation solution at a concentration of 5-30^106 cells/ml. Cells were aseptically filled and cryopreserved as PLX-C.
Example 3
Comparison of the Adherent Cells of WO/20071108003 (PLX) to the Adherent Cells of the Present Invention
Adherent cells produced by WO/2007/108003 (designated herein as PLX), as described in Example 1 hereinabove, were compared to the new adherent cells of the present invention (designated herein as PLX-C).
Materials and Experimental Methods
Cell Cycle analysis—PLX-C cells obtained by Celligen and PLX cells obtained by Plurix were fixed with 70% EtOH O.N, centrifuged and re-suspended in a Propidium Iodide (PI) solution containing 2 μg/ml PI (Sigma), 0.2 mg/ml Rnase A (Sigma) and 0.1% (v/v) Triton (Sigma) for 30 minutes. Cell cycle was analyzed by FACS.
Gene expression array (Microarray)—Adherent cells were obtained from human full term placentas and were expanded Plurix or by Celligen. Three different batches of cells were obtained from each of the expansion methods for further examination.
RNA was extracted from the cells (Qiagen-Rneasy micro kit) and applied to an Affymetrix whole genome expression array. The chip used GeneChip® Human Exon 1.0 ST Array (Affymetrix, Santa Clara, Calif., USA).
FACS analysis of membrane markers—cells were stained with monoclonal antibodies as previously described. In short, 400,000-600,000 cells were suspended in 0.1 ml flow cytometer buffer in a 5 ml test tube and incubated for 15minutes at room temperature (RT), in the dark, with each of the following monoclonal antibodies (MAbs): FITC-conjugated anti-human CD29 MAb (eBioscience), PE conjugated anti human CD73 MAb (Becton Dickinson), PE conjugated anti human CD105 MAb (eBioscience), PE conjugated anti human CD90 MAb (Becton Dickinson), FITC-conjugated anti-human CD45 MAb (IQProducts), PE-conjugated anti-human CD19 MAb (IQProducts), PE conjugated anti human CD14 MAb (IQProducts), FITC conjugated anti human HLA-DR MAb (IQProduct), PE conjugated anti human CD34 MAb (IQProducts), FITC conjugated anti human CD31 MAb (eBioscience), FITC conjugated anti human KDR MAb (R&D systems), anti human fibroblasts marker (D7-FIB) MAb(ACRIS), FITC-conjugated anti-human CD80 MAb (BD), FITC-conjugated anti-human CD86 MAb (BD), PE conjugated anti-human CD200 MAb (BD), FITC-conjugated anti-human CD40 MAb (BD), FITC-conjugated anti-human HLA-ABC MAb (BD), Isotype IgG1 FITC conjugated (IQ Products), Isotype IgG1 PE conjugated (IQ Products).
Cells were washed twice with flow cytometer buffer, resuspended in 500 μl flow cytometer buffer and analyzed by flow cytometry using FC-500 Flow Cytometer (Beckman Coulter). Negative controls were prepared with relevant isotype fluorescence molecules.
Mixed Lymphocyte Reaction (MLR)
2×105 peripheral blood (PB) derived MNC (from donor A) were stimulated with equal amount of irradiated (3000 Rad) PB derived MNCs (from donor B). Increasing amounts of PLX-Cs were added to the cultures. Three replicates of each group were seeded in 96-well plates. Cells were cultured in RPMI 1640 medium containing 20% FBS. Plates were pulsed with 1 μC 3H-thymidine during the last 18 hrs of the 5-day culturing. Cells were harvested over a fiberglass filter and thymidine uptake was quantified with scintillation counter.
For CFSE staining, PB-MNC cells were stained for CFSE (Molecular Probes) for proliferation measurement before culturing. Cells were collected after 5 days and the intensity of CFSE staining was detected by Flow Cytometry.
ELISA
ELISA was carried out as was previously described. In short, MNCs (isolated from peripheral blood) were stimulated with 5 μg/ml ConA (Sigma), 0.5 μg/ml LPS (SIGMA), or 10 μg/ml PHA (SIGMA) in the presence of PLX-C under humidified 5% CO2 atmosphere at 37° C. Supernatants were collected and subjected to cytokine analysis using ELISA kits for IFNγ (DIACLONE), TNFα (DIACLONE) and IL-10 (DIACLONE).
Experimental Results
The changes in manufacturing with Celligen as compared to Plurix resulted in several major differences (summarized in Table 1, below).







TABLE 1







Comparison between Plurix system (WO/2007/108003) and


Celligen system (teachings of the present invention)












Teachings of





the present



Parameter
WO/2007/108003
invention
Improvement





Working
280
1500
Scale up of the


volume (ml)


process.





Higher





production level





in the present





teachings (2-8





population





doubling)


Weight of
 1.4
 30
Scale up of the


carrier (gr)


process in the





present teachings.


Bed
Conic, 50 ml
Cylinder
The present


configuration
column
Packed bed
teachings --





Better flow of





medium and





nutrients.





WO/2007/108003 -





Inefficient





flow due to





narrow outlet





form the conic





structure





Better





homogeneity of





medium flow.





Channeling in the





plurix


Cell
3 × 106 cell/gr
5 × 106 cell/gr
Better cell to cell


concentration
carrier
carrier
interaction in the


at seeding


present teachings


(cell/gr





carrier)





Cell
0.015 × 106
0.1 × 106
Better cell to cell


concentration
cell/ml
cell/ml
interaction in the


at seeding


present teachings


(cell/ml)





Seeding
Seeding at low
Seeding at the
WO/2007/108003 -


procedure
medium volume
final working
Heterogenic



for 24 h followed
volume while
distribution of the



by addition of
agitating
cell culture inside



medium to final

the carrier bed



working volume

Insufficient





medium volume





in the first 24 h of





the run. Leading





to unsuitable





working





conditions (acidic





environment)


Production
14-21 days
4-12 days
Better product


phase


quality.


duration


Efficient harvest





process.





Better yield.





Lower cost





process in the





present teachings


Mode of
Repeated batch -
Perfusion
Present teachings -


operation
medium change
mode - rate
Moderate



twice a week
was adjusted
changes of the




according to
conditions




the glucose
regarding




concentration
medium




(the medium
composition




was changed
throughout the




at glucose
run




concentration
Continuous




of 550 ±
removal of toxic




50 mg/L)
agents produced





by the cells.





In batch mode -





lower





concentration of





essential





nutrients





(limiting factors)





Less cell debris


Harvest
Harvesting in
Harvesting
Present teachings -


procedure
50 ml tubes
inside the
More efficient



Trypsinization 3
bioreactor
process



cycles
Trypsinization
Harvest is carried




1 cycle
out in a close





system.





1 trypsinization





cycle - better





quality of the





cells.


Agitation
medium
Cell lift
Present teachings -



Circulation
impeller
Medium is



between reservoir

flowing through



container to the

the packed bed -



column using

Better supply of



peristaltic pump

nutrients and





oxygen to the





culture.





Homogeneity of





the medium





Improves other





control loops





(temp., DO, pH)


Temperature
The production
On-line direct
Present teachings -


control
was carried out
control.
more accurate



inside an
Heat transfer
measurement of



incubator.
via water
the culture



Indirect
jacket.
temperature.



temperature

Quick response.



control (of the

Short time to



incubator

reach set point.



chamber).





Heat transfer via





air interface




Temperature
Manually.
On-line direct
Present teachings -


monitoring
Indirect water
monitoring.
Better



temperature

monitoring and



monitoring.

control of the





process.





Quick response to





malfunctions.


DO
None
On-line
Present teachings -


monitoring

monitoring
Better





monitoring and





control of the





process.





Quick response to





malfunctions


DO control
None.
On-line direct
Present teachings -



Introduction of air
control of a
Better control



only
specific set
of DO level.




point using
Better




Air, O2 and
maintenance of a




N2.
specified working





conditions


pH
Only visual
On-line
Present teachings -


monitoring
monitoring
Control and
Better control


and control
(Phenol red as
monitoring
of pH level.



part of the

Better



medium)

maintenance of a





specified working





conditions


Aeration
Sparge only
Overlay
WO/2007/108003 -




(sparge as an
Aeration by




option)
sparge creates





foam that might





damage the cells.









The changes in the manufacturing process resulted in changes in characteristics of the obtained adherent cells. These differences are summarized below.
Cell cycle analysis of PLX manufactured by Plurix compared to PLX-C manufactured by Celligen—PLX-C cells obtained by Celligen were compared to PLX cells obtained by Plurix in order to examine the distribution of the cells between the different phases of the cell cycle. As is clear from FIGS. 3A-B, PLX-C cells expanded by Celligen exhibited typical proliferating profile (distribution of cells between the different phases of cell cycle). Specifically, 28% of cells were in S and G2/M phases (FIG. 3A). These results indicated that cells were harvested during proliferation and that the Celligen bioreactor conditions supported cell growth.
Microarray comparison between Plurix and Celligen obtained cells—gene expression arrays enabled to simultaneously monitor genome-wide expression profiles of adherent cells derived from human full term placentas expanded by Plurix (PLX) or by Celligen (PLX-C). These results enabled to asses the molecular mechanism underlying phenotypic variation between cells obtained by these different growth methods (see Table 2, below).







TABLE 2







Gene expression in Plurix cells (WO/2007/108003) compared to


Celligen cells (teachings of the present invention)










Celligen vs Plurix



Gene
(fold change)
p-value (treat)












interferon-induced protein with tetratricopeptide
17.52
0.0401812


repeats




aldehyde dehydrogenase 1 family, member A1
16.76
0.00145807


leukocyte-derived arginine aminopeptidase
13.99
3.88E−06


keratin 27 pseudogene 27
12.25
0.000224998


similar to Keratin, type I cytoskeletal 18 (Cytokerati
11.83
0.000304949


G protein-coupled receptor, family C, group 5,
10.35
3.39E−05


member A




integrin, alpha 6
9.84
0.0411667


G protein-coupled receptor 126
8.73
0.00197635


coagulation factor III (thromboplastin, tissue factor)
7.36
0.012192


Rho GDP dissociation inhibitor (GDI) beta
7.36
0.00200066


signal peptide, CUB domain, EGF-like 3
7.20
0.0255115


interferon-induced protein with tetratricopeptide
7.09
0.0139777


repeats




dickkopf homolog 1 (Xenopus laevis)
7.06
3.06E−07


NAD(P)H dehydrogenase, quinone 1
6.63
0.000282423


keratin 18
6.46
0.000514523


opioid growth factor receptor-like 1
5.96
0.00114551


mal, T-cell differentiation protein-like
5.95
0.00664216


neurofilament, medium polypeptide 150 kDa
5.86
0.0190611


DEP domain containing 1
5.82
0.000370513


cathepsin C
5.72
0.00532262


WAS
5.47
0.00178153


serpin peptidase inhibitor, clade B (ovalbumin),
5.44
0.0190218


member




solute carrier family 7, (cationic amino acid transporte
5.33
0.00688017


interferon-induced protein with tetratricopeptide repea
5.18
0.00357376


NUF2, NDC80 kinetochore complex component,
5.05
0.00276524


homolog (S. cere




SHC SH2-domain binding protein 1
4.95
0.00430878


thioredoxin reductase 1
4.86
0.000197486


lung cancer metastasis-associated protein
4.85
0.00148024


Rho GTPase activating protein 29
4.85
0.0466211


cell division cycle 20 homolog (S. cerevisiae)
4.80
0.00514206


family with sequence similarity 111, member B
4.63
0.000125819


PDZ binding kinase
4.54
0.00784983


establishment of cohesion 1 homolog 2 (S. cerevisiae)
4.53
0.000773033


guanylate binding protein 4
4.47
0.000215944


lipase A, lysosomal acid, cholesterol esterase
4.42
0.0167385


(Wolman dise




kinesin family member 20A
4.39
0.00582352


KIAA0101
4.28
0.0105909


cyclin-dependent kinase inhibitor 3 (CDK2-associated
4.25
0.000732492


dual




thymidylate synthetase
4.23
0.00685584


chromosome 13 open reading frame 3
4.18
0.000548296


aurora kinase A
4.16
0.00632571


nei endonuclease VIII-like 3 (E. coli)
4.14
0.00115606


centrosomal protein 55 kDa
4.13
0.0021952


oxidized low density lipoprotein (lectin-like) receptor 1
4.11
0.0205198


denticleless homolog (Drosophila)
4.05
0.00141153


anillin, actin binding protein
4.01
0.010923


ribonucleotide reductase M2 polypeptide
3.98
0.00834059


ankyrin repeat domain 1 (cardiac muscle)
3.93
0.00911953


transcription factor 19 (SC1)
3.89
0.00109627


keratin 18
3.89
0.000112551


non-SMC condensin I complex, subunit G
3.88
0.00537097


cyclin E2
3.87
0.000203389


trypsinogen C
3.86
0.00416276


small nucleolar RNA, C
3.81
0.0334484


tight junction protein 2 (zona occludens 2)
3.81
0.00012562


kinesin family member 18A
3.78
0.00134108


kinesin family member 2C
3.77
0.0059888


shugoshin-like 1 (S. pombe)
3.76
0.00101318


polo-like kinase 1 (Drosophila)
3.75
0.0140309


thymidine kinase 1, soluble
3.73
0.00124134


transcription factor 19 (SC1)
3.73
0.00124327


transcription factor 19 (SC1)
3.73
0.00124327


claspin homolog (Xenopus laevis)
3.71
0.00683624


GINS complex subunit 1 (Psf1 homolog)
3.69
0.00104515


microsomal glutathione S-transferase 1
3.67
0.041701


arylacetamide deacetylase-like 1
3.67
0.000902645


SPC25, NDC80 kinetochore complex component,
3.65
0.00568662


homolog (S. ce




integrin, alpha 4 (antigen CD49D, alpha 4 subunit of
3.62
0.0158411


VLA-4




catenin (cadherin-associated protein), alpha-like 1
3.57
7.46E−05


discs, large homolog 7 (Drosophila)
3.56
0.0317074


v-myb myeloblastosis viral oncogene homolog
3.55
0.0043878


(avian)-lik




serglycin
3.54
0.0443487


centromere protein N
3.53
0.000540143


cyclin A2
3.53
0.00965934


heat shock 22 kDa protein 8
3.52
0.0219583


sema domain, immunoglobulin domain (Ig), short
3.49
0.008548


basic doma




Rho GTPase activating protein 11A
3.49
0.00834174


Fanconi anemia, complementation group I
3.43
0.00464532


BUB1 budding uninhibited by benzimidazoles 1
3.42
0.0108258


homolog (yeast




ovary-specific acidic protein
3.42
0.00334641


cholinergic receptor, muscarinic 2
3.41
0.0320078


cell division cycle 2, G1 to S and G2 to M
3.41
0.0017111


protein regulator of cytokinesis 1
3.39
0.0325664


minichromosome maintenance complex component 5
3.38
0.00475504


sperm associated antigen 5
3.37
0.00906321


maternal embryonic leucine zipper kinase
3.34
0.00908391


small nucleolar RNA, C
3.33
0.0298703


carnitine palmitoyltransferase 1A (liver)
3.33
0.00170894


similar to Ubiquitin-conjugating enzyme E2S (Ubiqui
3.33
0.000415822


kinesin family member 11
3.33
0.00915145


NIMA (never in mitosis gene a)-related kinase 7
3.33
0.00159114


ADAM metallopeptidase with thrombospondin type 1
3.32
0.0102751


motif,




transforming, acidic coiled-coil containing protein 3
3.31
0.0014577


cyclin B1
3.29
0.0103092


MAD2 mitotic arrest deficient-like 1 (yeast)
3.28
0.00488102


dihydrofolate reductase
3.28
0.00178879


NIPA-like domain containing 3
3.27
0.00164708


cell division cycle associated 2
3.26
0.0122226


apolipoprotein B mRNA editing enzyme, catalytic
3.26
0.00308692


polypep




cyclin B2
3.25
0.016544


endonuclease domain containing 1
3.24
0.000429245


dihydrofolate reductase pseudogene
3.23
0.00141306


ATPase, Na+
3.23
0.000381464


replication factor C (activator 1) 3, 38 kDa
3.23
0.00109668


WD repeat domain 76
3.22
0.0023531


pleckstrin 2
3.17
0.0304429


Rac GTPase activating protein 1
3.17
0.00381613


PHD finger protein 19
3.17
0.000177604


deleted in lymphocytic leukemia, 2
3.15
0.0109528


centromere protein I
3.15
0.0106816


BRCA1 associated RING domain 1
3.14
0.000540414


regulator of G-protein signalling 4
3.13
0.00781061


STAM binding protein-like 1
3.11
0.0181743


sulfiredoxin 1 homolog (S. cerevisiae)
3.10
5.14E−05


chromosome 15 open reading frame 23
3.08
0.000147331


TTK protein kinase
3.08
0.0112171


non-SMC condensin II complex, subunit G2
3.08
0.0130322


villin 2 (ezrin)
3.07
0.0131934


stomatin
3.06
0.00387095


protein tyrosine phosphatase-like A domain containing
3.06
0.0419644


serpin peptidase inhibitor, clade B (ovalbumin),
3.05
0.0030439


member




kinesin family member 4A
3.05
0.0114203


hypothetical protein DKFZp762E1312
3.05
0.00726778


ubiquitin-conjugating enzyme E2S
3.04
0.00118205


hydroxysteroid dehydrogenase like 2
3.03
3.71E−05


ATPase family, AAA domain containing 2
3.01
0.00415258


TPX2, microtubule-associated, homolog (Xenopus
3.00
0.0253137


laevis)




histone cluster 1, H4d
3.00
0.030183


kinesin family member 23
2.99
0.00790585


heat shock 70 kDa protein 2
2.99
0.0215102


origin recognition complex, subunit 1-like (yeast)
2.99
0.00207753


dihydrofolate reductase
2.98
0.00307793


hyaluronan-mediated motility receptor (RHAMM)
2.97
0.00467816


3′-phosphoadenosine 5′-phosphosulfate synthase 2
2.97
1.43E−05


glycerol-3-phosphate dehydrogenase 2 (mitochondrial)
2.95
0.00211969


nucleolar and spindle associated protein 1
2.95
0.00520875


diaphanous homolog 3 (Drosophila)
2.95
0.00107709


kinesin family member 14
2.94
0.00947901


histone cluster 1, H1b
2.93
0.0470898


guanine nucleotide binding protein (G protein), alpha
2.92
0.00184597


inhi




minichromosome maintenance complex component 8
2.92
0.000841489


cancer susceptibility candidate 5
2.92
0.0330594


leukotriene B4 12-hydroxydehydrogenase
2.92
0.000685452


glutamate-cysteine ligase, modifier subunit
2.91
0.00378868


forkhead box M1
2.91
0.0203154


adipose differentiation-related protein
2.90
0.000331751


membrane bound O-acyltransferase domain containing 1
2.90
0.01185


ubiquitin-conjugating enzyme E2T (putative)
2.90
0.00741886


cell division cycle associated 3
2.89
0.006289


integrin, alpha 3 (antigen CD49C, alpha 3 subunit of
2.88
0.00574148


VLA-3




coagulation factor XIII, B polypeptide
2.88
0.0294465


RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)
2.87
0.000854739


ATP-binding cassette, sub-family C (CFTR
2.87
0.00382491


family with sequence similarity 29, member A
2.85
0.00111165


SH2 domain containing 4A
2.84
0.0323646


membrane protein, palmitoylated 1, 55 kDa
2.84
0.000396285


CDC28 protein kinase regulatory subunit 1B
2.84
0.0107391


PSMC3 interacting protein
2.84
0.00766442


elastin microfibril interfacer 2
2.84
0.0192072


topoisomerase (DNA) II alpha 170 kDa
2.83
0.0321109


transmembrane protein 106C
2.82
0.000214223


histone cluster 1, H3b
2.80
0.0304598


chromosome 18 open reading frame 24
2.80
0.00347442


epidermal growth factor receptor pathway substrate 8
2.79
0.0194949


high-mobility group nucleosomal binding domain 2
2.78
0.0030536


SCL
2.78
0.00390288


hect domain and RLD 4
2.78
0.00679184


ASF1 anti-silencing function 1 homolog B (S. cerevisiae)
2.77
0.00543408


thyroid hormone receptor interactor 13
2.76
0.0118319


cell division cycle associated 8
2.75
0.00619878


kinesin family member C1
2.74
0.00821937


high-mobility group nucleosomal binding domain 2
2.73
0.00384071


ornithine decarboxylase 1
2.73
0.00144868


v-myb myeloblastosis viral oncogene homolog
2.71
0.00989416


(avian)-like 2




KIT ligand
2.70
0.00641955


dual-specificity tyrosine-(Y)-phosphorylation
2.70
0.0234606


regulated ki




intraflagellar transport 80 homolog (Chlamydomonas)
2.70
0.0247286


transmembrane protein 48
2.69
0.00458248


EBNA1 binding protein 2
2.69
0.00296292


ZW10 interactor
2.69
1.88E−05


exonuclease 1
2.68
0.00739393


transketolase (Wernicke-Korsakoff syndrome)
2.68
1.92E−05


somatostatin receptor 1
2.68
0.0144901


isocitrate dehydrogenase 3 (NAD+) alpha
2.67
0.00297129


cytoskeleton associated protein 2
2.67
0.0030499


minichromosome maintenance complex component 4
2.67
0.00342054


inhibitor of DNA binding 1, dominant negative helix-
2.66
0.036485


loop-hel




CDC28 protein kinase regulatory subunit 1B
2.66
0.0145263


keratin 18
2.66
8.40E−05


CD97 molecule
2.66
0.00994045


chromosome 6 open reading frame 173
2.64
0.00222408


BTB (POZ) domain containing 3
2.62
0.0166824


deafness, autosomal dominant 5
2.62
0.00235481


KIAA0286 protein
2.62
0.00130563


Fanconi anemia, complementation group D2
2.61
0.0281405


polo-like kinase 4 (Drosophila)
2.60
0.00209633


ribonucleotide reductase M1 polypeptide
2.60
0.000170076


malic enzyme 1, NADP(+)-dependent, cytosolic
2.59
0.0435444


non-SMC condensin I complex, subunit H
2.59
0.0216752


S100 calcium binding protein A3
2.58
0.0324073


ubiquitin-conjugating enzyme E2L 3
2.57
0.00343347


BUB1 budding uninhibited by benzimidazoles 1
2.56
0.0166047


homolog beta




glycerol kinase
2.55
2.66E−05


TAF9B RNA polymerase II, TATA box binding
2.54
0.0170365


protein (TBP)-as




TAF9B RNA polymerase II, TATA box binding
2.54
0.0170365


protein (TBP)-as




histone cluster 1, H2bg
2.52
0.000180822


high-mobility group box 2
2.52
0.0196872


NIMA (never in mitosis gene a)-related kinase 2
2.50
0.00289469


proline rich 11
2.50
0.0357125


myopalladin
2.49
0.0255088


brix domain containing 1
2.49
0.00471977


cell division cycle associated 5
2.49
0.01021


fucosidase, alpha-L-2, plasma
2.49
0.00540929


cyclin-dependent kinase 2
2.49
0.00250724


lamin B receptor
2.49
0.000151784


hypoxanthine phosphoribosyltransferase 1 (Lesch-
2.49
0.000634057


Nyhan synd




tripartite motif-containing 25
2.47
0.0456344


proteasome (prosome, macropain) subunit, beta type, 9
2.46
0.0202595


(lar




proteasome (prosome, macropain) subunit, beta type, 9
2.46
0.0202595


(lar




proteasome (prosome, macropain) subunit, beta type, 9
2.46
0.0202595


(lar




sphingomyelin synthase 2
2.46
0.0020701


transmembrane protein 62
2.45
0.00761064


glucose-6-phosphate dehydrogenase
2.44
0.00278311


PHD finger protein 1
2.44
0.010191


retinoblastoma-like 1 (p107)
2.44
0.00319946


KIAA1524
2.43
0.0380688


ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-
2.43
0.00830766


1,




cofilin 2 (muscle)
2.43
0.0459235


hypothetical protein LOC201725
2.42
0.000313319


cell division cycle 25 homolog A (S. pombe)
2.42
0.000341692


breast cancer 1, early onset
2.41
0.0180553


transaldolase 1
2.41
0.00199537


mRNA turnover 4 homolog (S. cerevisiae)
2.41
0.00373104


glucosaminyl (N-acetyl) transferase 1, core 2 (beta-
2.41
0.0197148


1,6-N-




cysteine rich transmembrane BMP regulator 1
2.41
0.0267286


(chordin-like)




tissue factor pathway inhibitor (lipoprotein-associated
2.40
0.0356227


chromosome 16 open reading frame 59
2.40
0.00185191


glycogenin 1
2.39
0.0224317


transmembrane protein 154
2.39
0.0045589


tubulointerstitial nephritis antigen-like 1
2.39
0.00510812


CTP synthase
2.38
8.80E−05


phenylalanyl-tRNA synthetase, beta subunit
2.38
0.000245973


geminin, DNA replication inhibitor
2.38
0.00167629


lamin B1
2.37
0.0477748


SPC24, NDC80 kinetochore complex component,
2.36
0.00287227


homolog (S. ce




glutathione reductase
2.36
0.00353875


ribosomal protein L22-like 1
2.36
0.00335381


fumarylacetoacetate hydrolase (fumarylacetoacetase)
2.36
3.88E−05


small nucleolar RNA, C
2.35
0.0188991


family with sequence similarity 64, member A
2.35
0.0019785


epithelial cell transforming sequence 2 oncogene
2.35
0.000571152


polymerase (DNA directed), epsilon 2 (p59 subunit)
2.34
0.00479612


glycerol kinase
2.34
3.37E−06


glutathione S-transferase M2 (muscle)
2.33
0.0402076


elongation factor, RNA polymerase II, 2
2.33
0.0130017


thioredoxin
2.33
0.009636


polymerase (DNA directed), alpha 2 (70 kD subunit)
2.32
0.0033903


breast cancer 2, early onset
2.32
0.00586847


CDC45 cell division cycle 45-like (S. cerevisiae)
2.32
0.00735977


H2A histone family, member Z
2.32
0.0129697


transporter 1, ATP-binding cassette, sub-family B
2.31
0.0164234


(MDR




transporter 1, ATP-binding cassette, sub-family B
2.31
0.0164234


(MDR




transporter 1, ATP-binding cassette, sub-family B
2.31
0.0164234


(MDR




nucleolar complex associated 3 homolog (S. cerevisiae)
2.30
0.000373346


ATPase, Ca++ transporting, plasma membrane 4
2.30
0.023011


minichromosome maintenance complex component 7
2.30
0.0457691


TIMELESS interacting protein
2.29
0.00771062


von Hippel-Lindau binding protein 1
2.28
0.00329061


ras-related C3 botulinum toxin substrate 2 (rho family,
2.28
0.0292466


sma




thymopoietin
2.28
0.0223176


peptidylprolyl isomerase F (cyclophilin F)
2.28
0.00093846


activated leukocyte cell adhesion molecule
2.27
0.00242163


polycomb group ring finger 5
2.27
0.000294142


Ran GTPase activating protein 1
2.27
9.68E−05


replication factor C (activator 1) 4, 37 kDa
2.26
0.00164152


tubulin, beta 2C
2.26
0.000346744


minichromosome maintenance complex component 10
2.26
0.0037925


H2B histone family, member S
2.25
0.000885505


gamma-glutamyl hydrolase (conjugase,
2.25
0.0195219


folylpolygammaglutamyl




transcription termination factor, RNA polymerase II
2.25
0.000393489


polymerase (DNA directed), delta 2, regulatory
2.25
0.0123823


subunit 50k




transporter 1, ATP-binding cassette, sub-family B
2.25
0.00859077


(MDR




transporter 1, ATP-binding cassette, sub-family B
2.25
0.00859077


(MDR




transporter 1, ATP-binding cassette, sub-family B
2.25
0.00859077


(MDR




histone cluster 1, H2bf
2.25
0.0124279


eukaryotic translation initiation factor 1A, X-linked
2.24
0.00330183


phosphoglucomutase 2
2.24
0.00818204


peroxisomal D3,D2-enoyl-CoA isomerase
2.24
0.00148722


interferon-induced protein with tetratricopeptide
2.24
0.0177928


repeats




G-2 and S-phase expressed 1
2.23
0.0241887


minichromosome maintenance complex component 2
2.23
0.0021347


family with sequence similarity 72, member A
2.23
0.00143248


RMI1, RecQ mediated genome instability 1, homolog
2.23
0.00294705


(S. cerev




FLJ20105 protein
2.23
0.0127979


multiple coagulation factor deficiency 2
2.22
0.0116892


phytoceramidase, alkaline
2.22
0.0157729


coiled-coil domain containing 68
2.22
0.00227586


dedicator of cytokinesis 11
2.21
0.00697577


platelet-derived growth factor alpha polypeptide
2.21
0.00176418


N-acylsphingosine amidohydrolase (non-lysosomal
2.20
0.00728536


cerami




S-phase kinase-associated protein 2 (p45)
2.20
0.00230153


polymerase (RNA) III (DNA directed) polypeptide G
2.20
0.0298794


(32 kD)




ADP-ribosylation factor-like 6 interacting protein 1
2.20
0.00139745


histone cluster 1, H2bh
2.19
0.0377748


origin recognition complex, subunit 5-like (yeast)
2.19
0.049697


CDC28 protein kinase regulatory subunit 2
2.19
0.0128024


histone cluster 1, H4c
2.19
0.0112695


hypothetical protein LOC729012
2.19
0.000446087


DEAD (Asp-Glu-Ala-Asp) box polypeptide 39
2.19
0.000340561


chromatin assembly factor 1, subunit B (p60)
2.18
0.0119687


MLF1 interacting protein
2.18
0.0177203


microtubule associated serine
2.18
0.00536974


MHC class I polypeptide-related sequence B
2.18
0.0165406


shugoshin-like 2 (S. pombe)
2.18
0.000852557


COP9 constitutive photomorphogenic homolog
2.18
0.000793512


subunit 6 (Arab




methylenetetrahydrofolate dehydrogenase (NADP+
2.18
0.00119726


dependent)




chromosome 6 open reading frame 167
2.18
0.0011095


pituitary tumor-transforming 1
2.17
0.0485166


ribonuclease H2, subunit A
2.17
0.00669936


X-ray repair complementing defective repair in
2.16
0.0369865


Chinese ham




membrane protein, palmitoylated 5 (MAGUK p55
2.16
0.00211873


subfamily memb




karyopherin alpha 2 (RAG cohort 1, importin alpha 1)
2.16
0.000650645


pleckstrin homology domain containing, family A
2.15
0.0256434


(phosphoi




ribosomal protein L39-like
2.15
0.00429384


karyopherin alpha 2 (RAG cohort 1, importin alpha 1)
2.15
0.000700649


amyloid beta (A4) precursor protein-binding, family
2.15
0.00201004


B, m




minichromosome maintenance complex component 3
2.14
0.0018389


histone cluster 1, H2ai
2.14
0.0129155


chromosome 13 open reading frame 34
2.14
0.000702936


RAD18 homolog (S. cerevisiae)
2.14
0.0016685


WD repeat and HMG-box DNA binding protein 1
2.13
0.0034833


sulfide quinone reductase-like (yeast)
2.13
0.0473641


chromosome 16 open reading frame 63
2.12
0.000804179


M-phase phosphoprotein 1
2.12
0.0271814


minichromosome maintenance complex component 6
2.12
0.0161279


homeobox A9
2.11
0.00520942


fibroblast growth factor 9 (glia-activating factor)
2.10
0.0475844


cell division cycle 25 homolog C (S. pombe)
2.10
0.0169914


chromosome 9 open reading frame 64
2.10
0.0265979


U2AF homology motif (UHM) kinase 1
2.09
0.0255167


replication factor C (activator 1) 2, 40 kDa
2.09
0.00768959


hypothetical protein LOC440894
2.09
0.0103358


small nuclear ribonucleoprotein D1 polypeptide
2.09
0.0334665


16 kDa




CSE1 chromosome segregation 1-like (yeast)
2.09
0.0013662


phosphatidylinositol glycan anchor biosynthesis, class W
2.09
0.0151967


centromere protein O
2.09
0.00397056


family with sequence similarity 20, member B
2.09
0.00460031


hypothetical protein FLJ40869
2.09
0.00444509


guanine nucleotide binding protein (G protein),
2.08
0.00140559


gamma 11




calcyclin binding protein
2.08
0.00524566


ATP-binding cassette, sub-family E (OABP), member 1
2.08
0.00454751


CD44 molecule (Indian blood group)
2.08
0.000651436


exosome component 8
2.08
0.00132017


family with sequence similarity 102, member B
2.08
0.025743


histone cluster 2, H3d
2.07
0.0102932


family with sequence similarity 33, member A
2.07
0.000318673


Fanconi anemia, complementation group B
2.07
0.000255109


kinesin family member 22
2.07
0.0192406


histone cluster 1, H2ai
2.07
0.0161621


vaccinia related kinase 1
2.06
0.0233182


integrator complex subunit 7
2.06
0.000841371


flap structure-specific endonuclease 1
2.06
0.006882


hypothetical protein FLJ25416
2.06
0.000177531


ecotropic viral integration site 2B
2.06
0.0171408


retinitis pigmentosa 2 (X-linked recessive)
2.05
0.0264185


centromere protein L
2.05
0.000880856


cofactor required for Sp1 transcriptional activation,
2.04
0.00141809


subu




chromosome 20 open reading frame 121
2.04
0.0146323


family with sequence similarity 72, member A
2.04
0.00162905


family with sequence similarity 72, member A
2.04
0.00165234


eukaryotic translation initiation factor 1A, X-linked
2.04
0.00520549


elongation factor, RNA polymerase II, 2
2.03
0.0458007


ATPase, Na+
2.03
0.0189108


histone cluster 1, H3a
2.03
0.0244273


brix domain containing 1
2.03
0.00981178


sushi domain containing 1
2.03
0.0258164


ectonucleoside triphosphate diphosphohydrolase 6
2.03
0.00423628


(putativ




fructosamine 3 kinase
2.03
0.00470972


Bloom syndrome
2.02
0.0209259


tubulin, alpha 1c
2.01
0.00862586


E2F transcription factor 2
2.01
0.0496479


exosome component 2
2.01
0.00649147


kinesin family member 22
2.01
0.0242075


LTV1 homolog (S. cerevisiae)
2.01
0.00812652


dihydrolipoamide S-acetyltransferase (E2 component
2.01
0.00179011


of pyruv




v-ral simian leukemia viral oncogene homolog B (ras
2.01
0.012225


related




ring finger and WD repeat domain 3
2.01
0.0013797


annexin A1
2.01
0.0173578


elaC homolog 2 (E. coli)
2.00
0.00266504


aldehyde dehydrogenase 9 family, member A1
2.00
0.00911609


tubulin, alpha 4a
2.00
0.0435427


nuclear pore complex interacting protein
−2.00
0.00111223


oculomedin
−2.01
0.00778869


similar to PI-3-kinase-related kinase SMG-1
−2.01
0.0356628


golgi autoantigen, golgin subfamily a-like pseudogene
−2.01
0.00770626


spectrin repeat containing, nuclear envelope 1
−2.01
0.00438469


nuclear pore complex interacting protein
−2.01
0.00117582


sushi, nidogen and EGF-like domains 1
−2.01
0.00161129


integrin, alpha V (vitronectin receptor, alpha
−2.02
0.00252702


polypeptide




cyclin-dependent kinase inhibitor 2B (p15, inhibits
−2.04
0.0150268


CDK4)




lysyl oxidase-like 4
−2.04
0.0120148


nuclear pore complex interacting protein
−2.04
0.000213956


calcium
−2.04
0.00657494


calsyntenin 3
−2.04
0.00300887


cell adhesion molecule 1
−2.05
0.0261129


solute carrier family 22 (organic cation transporter),
−2.05
0.0137275


RUN and FYVE domain containing 3
−2.05
0.00387265


glucosidase, alpha; acid (Pompe disease, glycogen
−2.05
0.000418401


storage di




nuclear pore complex interacting protein
−2.05
0.00988632


proline-rich nuclear receptor coactivator 1
−2.06
0.0039587


membrane metallo-endopeptidase
−2.06
0.0152684


PHD finger protein 21A
−2.06
0.00980401


Rho GTPase-activating protein
−2.06
0.00705186


homeobox B6
−2.06
0.00301714


nuclear pore complex interacting protein
−2.07
0.00032839


phospholipase A2 receptor 1, 180 kDa
−2.07
0.00069343


nuclear pore complex interacting protein
−2.08
0.000352007


slit homolog 3 (Drosophila)
−2.08
0.02844


nuclear pore complex interacting protein
−2.09
0.000414309


cyclin-dependent kinase 6
−2.09
0.0456892


dynamin 1
−2.09
0.00139674


jumonji, AT rich interactive domain 1B
−2.09
0.00861002


calcium binding and coiled-coil domain 1
−2.09
0.00370041


insulin-like growth factor 1 receptor
−2.09
0.00114467


nuclear pore complex interacting protein
−2.10
0.000377834


CD82 molecule
−2.10
0.0175517


bromodomain adjacent to zinc finger domain, 2B
−2.10
9.88E−05


—
−2.10
0.00666187


synaptotagmin XI
−2.11
0.0129428


KIAA1546
−2.11
0.000255634


jun B proto-oncogene
−2.12
0.0120169


CXXC finger 6
−2.12
0.0277527


nuclear pore complex interacting protein
−2.14
0.00282604


Cdon homolog (mouse)
−2.15
0.0350357


B-cell CLL
−2.15
0.00343507


nuclear pore complex interacting protein
−2.15
0.00263888


v-abl Abelson murine leukemia viral oncogene
−2.16
0.0136688


homolog 1




nuclear pore complex interacting protein
−2.16
0.00583397


FAT tumor suppressor homolog 1 (Drosophila)
−2.18
0.0158766


transformer-2 alpha
−2.18
0.012256


chimerin (chimaerin) 1
−2.18
0.0287031


milk fat globule-EGF factor 8 protein
−2.18
0.000987073


vitamin D (1,25-dihydroxyvitamin D3) receptor
−2.19
0.000192208


neuroblastoma, suppression of tumorigenicity 1
−2.20
0.00090639


jumonji domain containing 1A
−2.20
0.0188513


WNK lysine deficient protein kinase 1
−2.21
1.57E−05


protocadherin beta 14
−2.21
0.0103892


cortactin binding protein 2
−2.21
2.28E−05


WW domain containing transcription regulator 1
−2.22
0.0379899


cyclin L1
−2.22
0.00831474


nuclear factor of activated T-cells, cytoplasmic,
−2.22
0.00786451


calcine




pellino homolog 1 (Drosophila)
−2.23
0.00939357


golgi autoantigen, golgin subfamily a-like pseudogene
−2.24
0.00603583


chromosome 7 open reading frame 10
−2.26
0.00738442


golgi autoantigen, golgin subfamily a-like pseudogene
−2.27
0.00320764


small Cajal body-specific RNA 17
−2.27
0.0301336


latent transforming growth factor beta binding protein 2
−2.29
4.08E−05


golgi autoantigen, golgin subfamily a, 8A
−2.29
0.0111179


inhibin, beta A (activin A, activin AB alpha
−2.29
0.00877271


polypeptide)




solute carrier family 41, member 2
−2.30
0.00453672


forkhead box P1
−2.30
0.0463138


matrix metallopeptidase 14 (membrane-inserted)
−2.31
1.93E−05


transcription factor 4
−2.31
0.0367869


jun oncogene
−2.32
7.21E−05


neuroepithelial cell transforming gene 1
−2.33
0.0109689


asporin
−2.33
0.000659873


v-fos FBJ murine osteosarcoma viral oncogene
−2.35
0.0138624


homolog




ephrin-B2
−2.36
0.00611474


WD repeat and SOCS box-containing 1
−2.36
0.0387851


similar to dJ402H5.2 (novel protein similar to wo
−2.36
0.00621503


PX domain containing serine
−2.38
0.000927628


collagen, type VII, alpha 1 (epidermolysis bullosa,
−2.38
0.00109233


dystr




AE binding protein 1
−2.39
0.000105628


peroxidasin homolog (Drosophila)
−2.40
0.00219049


calcium channel, voltage-dependent, L type, alpha 1C
−2.41
0.0189661


sub




Prader-Willi syndrome chromosome region 1
−2.45
0.0415526


midline 1 (Opitz
−2.45
0.00130803


nuclear pore complex interacting protein
−2.45
0.00354416


chromosome 1 open reading frame 54
−2.47
0.0186089


transmembrane protein 16A
−2.48
0.0481085


basic helix-loop-helix domain containing, class B, 2
−2.49
0.00270257


nuclear pore complex interacting protein
−2.50
0.00316496


runt-related transcription factor 1 (acute myeloid
−2.50
0.000607387


leukemi




zinc finger protein 292
−2.50
0.029832


fibronectin leucine rich transmembrane protein 2
−2.51
0.0135122


nuclear pore complex interacting protein
−2.51
0.00283418


potassium voltage-gated channel, subfamily G,
−2.54
0.0244306


member 1




interleukin 19
−2.54
0.0310328


transforming growth factor, beta 3
−2.54
0.0287865


dihydropyrimidinase-like 3
−2.55
0.0165203


golgi autoantigen, golgin subfamily a, 8B
−2.56
0.0121417


hypothetical protein PRO2012
−2.57
0.00756704


SATB homeobox 2
−2.57
0.039781


t-complex 11 (mouse)-like 2
−2.57
0.0324227


ring finger protein 122
−2.57
0.0236621


chromosome 8 open reading frame 57
−2.59
0.00261522


ADAM metallopeptidase with thrombospondin type 1
−2.60
0.0113968


motif,




sushi, von Willebrand factor type A, EGF and
−2.63
2.23E−05


pentraxin dom




ST6 beta-galactosamide alpha-2,6-sialyltranferase 2
−2.64
0.0216987


sortilin-related VPS10 domain containing receptor 2
−2.65
0.00936311


protocadherin beta 9
−2.66
0.0285124


chromosome 5 open reading frame 13
−2.67
0.00410172


Enah
−2.68
0.0077547


pyridoxal-dependent decarboxylase domain containing 2
−2.69
0.00683647


similar to nuclear pore complex interacting protein
−2.70
0.0187322


nuclear pore complex interacting protein
−2.70
0.00368967


transmembrane protein 119
−2.70
0.00801387


chromosome 14 open reading frame 37
−2.70
0.0182453


sushi-repeat-containing protein, X-linked 2
−2.71
0.0253856


PDZ domain containing RING finger 3
−2.71
0.00931014


collagen, type XII, alpha 1
−2.72
0.000204664


matrix-remodelling associated 5
−2.72
0.000317637


collagen, type V, alpha 1
−2.72
0.0166427


dystrophin related protein 2
−2.72
0.0137557


ATP-binding cassette, sub-family A (ABC1), member 1
−2.73
0.00131361


trophinin
−2.77
0.00298044


cornichon homolog 3 (Drosophila)
−2.78
0.0261738


formin binding protein 1-like
−2.78
0.00290401


brain and acute leukemia, cytoplasmic
−2.78
0.0476919


protein tyrosine phosphatase, receptor type, U
−2.80
0.0270428


hypothetical protein MGC24103
−2.82
0.0346673


interferon induced with helicase C domain 1
−2.83
0.0024839


phospholipid transfer protein
−2.84
0.00999206


immediate early response 3
−2.87
0.0152127


immediate early response 3
−2.87
0.0152127


ADAM metallopeptidase domain 12 (meltrin alpha)
−2.87
0.000870288


synaptic vesicle glycoprotein 2A
−2.88
0.00704212


chromosome 9 open reading frame 3
−2.88
0.00410177


thioredoxin interacting protein
−2.90
0.0135494


early growth response 1
−2.93
0.000425035


small nucleolar RNA, C
−2.94
0.00666866


small nucleolar RNA, C
−2.95
0.00765575


immediate early response 3
−2.99
0.0167309


low density lipoprotein-related protein 1 (alpha-2-
−2.99
4.26E−05


macroglo




bicaudal C homolog 1 (Drosophila)
−2.99
0.0347162


homeobox B2
−3.03
0.00665994


small nucleolar RNA, C
−3.10
0.0274043


small nucleolar RNA, C
−3.10
0.0274043


matrix metallopeptidase 2 (gelatinase A, 72 kDa
−3.13
5.59E−05


gelatinase,




KIAA1641
−3.14
0.00659194


collagen, type VI, alpha 3
−3.14
2.09E−06


homeobox A2
−3.15
0.0435423


SH3 and PX domains 2B
−3.15
0.0244357


collagen, type VI, alpha 2
−3.16
0.0149554


chromosome 9 open reading frame 3
−3.21
0.0233723


small nucleolar RNA, C
−3.24
0.0104491


small nucleolar RNA, C
−3.24
0.0104491


—
−3.27
0.00488845


UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-
−3.35
0.00964109


acetylga




cholesterol 25-hydroxylase
−3.38
0.0445558


KIAA1641
−3.40
0.013175


ring finger protein 144
−3.40
0.0135334


versican
−3.41
0.023885


angiopoietin-like 2
−3.42
0.0245161


KIAA1641
−3.44
0.0170531


FBJ murine osteosarcoma viral oncogene homolog B
−3.54
0.00025573


similar to RIKEN cDNA 1110018M03
−3.59
0.00516476


early growth response 2 (Krox-20 homolog,
−3.62
0.00821813


Drosophila)




dachsous 1 (Drosophila)
−3.63
0.00697244


kinesin family member 26B
−3.64
0.00363199


distal-less homeobox 5
−3.66
0.000640157


similar to Protein KIAA0220
−3.69
0.0302619


insulin-like growth factor 1 receptor
−3.71
3.42E−05


protein tyrosine phosphatase, receptor type, N
−3.77
0.0294569


KIAA1641
−3.85
0.0191782


sushi-repeat-containing protein, X-linked
−3.85
0.00370941


microfibrillar-associated protein 2
−3.91
0.0152901


complement component 1, s subcomponent
−3.97
0.0395863


CD24 molecule
−3.99
0.0340122


homeobox B3
−4.02
0.0354368


trichorhinophalangeal syndrome I
−4.02
0.00557712


Kallmann syndrome 1 sequence
−4.04
0.000548703


leucine rich repeat containing 17
−4.09
0.0263961


plexin domain containing 2
−4.32
0.031799


PTK7 protein tyrosine kinase 7
−4.42
0.000116114


supervillin
−4.43
0.0412717


zinc finger protein 521
−4.58
0.00668815


calbindin 2, 29 kDa (calretinin)
−4.77
0.0290743


ras homolog gene family, member J
−4.79
0.00197982


integrin, alpha 11
−4.80
0.000390317


odz, odd Oz
−5.05
0.00172671


F-box protein 32
−5.52
0.0212957


raftlin family member 2
−5.72
0.0260454


clusterin
−5.74
0.0303973


neurotrimin
−5.79
3.78E−06


WNT1 inducible signaling pathway protein 1
−5.86
0.000672342


insulin-like growth factor binding protein 5
−6.34
0.011614


sulfatase 2
−6.34
5.88E−05


microfibrillar-associated protein 4
−6.93
0.00155578


junctional adhesion molecule 2
−7.07
0.0306758


fibronectin type III domain containing 1
−7.29
0.0334696


sarcoglycan, delta (35 kDa dystrophin-associated
−7.37
0.000881984


glycoprotei




hephaestin
−7.53
0.0123141


serpin peptidase inhibitor, clade F (alpha-2 antiplasmi
−7.66
0.00362941


cystatin SN
−7.96
0.0496433


hemicentin 1
−8.18
0.0461603


tenascin C (hexabrachion)
−8.32
8.26E−05


biglycan
−8.62
0.00161284


transmembrane, prostate androgen induced RNA
−11.20
0.000100935


carboxypeptidase E
−11.22
0.00738131









Expression of cellular markers on PLX-C cells—the surface antigens expressed by PLX-C were examined using monoclonal antibodies. Results indicated that PLX-C cells were characterized by the positive markers: CD73, CD29 and CD105 and the negative markers: CD34, CD45, CD19, CD14, CD200 and HLA-DR. In some experiments, the immune phenotype test specifications were set as: ≧90% for all positive markers and ≦3% for all negative markers.
Furthermore, as shown in FIGS. 4A-B, PLX-C cultures did not express endothelial markers as shown by negative staining for the two endothelial markers CD31 and KDR. However, PLX-C expression of a fibroblast-typical marker was evident (expression of D7-fib, FIG. 4C). In addition, as is shown in FIG. 4D, PLX-C cells negatively express CD200.
Immunogenecity and immunomodulatory properties of PLX-C cells—as PLX-C is comprised of adherent cells derived from placenta, it is expected to express HLA type I, which is expressed by all cells of the body and is known to induce an alloreactive immune response. HLA type II and other co-stimulatory molecules are typically expressed only on the surface of Antigen Presenting Cells (APCs).
In order to examine the immunogenicity of the obtained PLX-C cells, the expression of co-stimulatory molecules on the surface of these cell membranes were performed. FACS analysis demonstrated the absence of CD80, CD86 and CD40 on the PLX-C cell membranes (FIGS. 5A-C). Moreover, PLX-C expressed low levels HLA class I as detected by staining for HLA A/B/C (FIG. 5D). The expression of stimulatory and co-stimulatory molecules was similar to bone marrow (BM) derived MSCs (as shown in FIGS. 5A-D).
To further investigate the immunogenecity as well as the immunomodulation properties of PLX-C cells, Mix Lymphocyte Reaction (MLR) tests were performed. As shown in FIG. 6A-B, PLX-C cells both escape allorecognition and reduce T cell response, as measured by Thymidine incorporation. Furthermore, the reduction in lymphocytes proliferation (evaluated by CPM measurement) was higher as the number of PLX-C cells increased (in a dose dependent manner). PLX-C also reduced lymphocyte proliferation following mitogenic stimuli, such as Concavalin A (Con A, FIG. 6B) and Phytohemagglutinin (PHA), and non-specific stimulation by anti-CD3, anti-CD28 (data not shown).
In order to investigate the mechanism of action by which PLX-C immunomodulate lymphocyte proliferation, and to see if this action is mediated via cell to cell interaction or cytokines secretion, PB derived Mononuclear cells (MNCs) were stimulated by PHA using the transwell method (which prevents cell to cell contact but enables the diffusion of cytokines between the two compartments). Results showed that the inhibition of proliferation maintained even when cell to cell contact was inhibited (data not shown).
Cytokines secretion—as depicted hereinabove, PLX-C reduce the proliferation rate of lymphocytes, probably through soluble factors. Further investigation of the cytokines secreted by lymphocytes in response to PLX-C was performed to elucidate the mechanism of action of PLX-C. As depicted in FIGS. 7A-B, culturing of mononuclear cells with PLX-C slightly reduces the secretion of the pro-inflammatory cytokine INFγ and dramatically reduces the secretion of TNFα (even in the presence of low amounts of PLX-C). In addition, following lipopolysaccharide (LPS) stimulation, PB derived MNCs secretion of IL-10 increased in the presence of PLX-C, while the secretion level of TNFα decreased, in a dose dependent manner (FIG. 7C).
Example 4
Comparison of Osteocyte Differentiation of 2D Adherent Cells of the Present Invention and Bone Marrow Cells
The new adherent cells of the present invention (from placenta origin) were grown under osteocyte differentiation stimulating conditions at their 2D adherent cell stage in comparison to bone marrow derived cells.
Materials and Experimental Methods
Osteogenesis
Osteogenesis was carried out according to Chemicon osteogenesis kit (cat no. scr028, Millipore, Mass., USA)
Osteogenesis Induction Medium
Osteogenesis induction medium was freshly made prior to each medium exchange using the kit components (See Table 3, below).







TABLE 3







Osteogenesis medium components











Stock




Component
concentration
Amount
Final con














DMEM low glucose

8.7
ml
87%


(Invitrogen, Gibco)






Serum (heat inactivated)

1
ml
10%












dexamethasone
1 mM
1
μl
0.1
μM


Asorbic Acid-2-Phosphate
0.1M
20
μl
0.2
mM


solution







Glycerol-2-Phosphate Solution
  1M
100
μL
10
Mm











L-glutamine
×100
100
μl
×1


Pen & Strep
×100
100
μl
×1









To arrive at 1 mM dexamethasone solution, 900 μl ethanol was added to 100 μl dexamethasone 10 mM solution. Stock solution was stored with the rest of the kit's components at −20° C. A 50 ml serum vial was heat inactivated, divided into 5 ml aliquots and kept at −20° C. until use.
Coating 24-well Tissue Culture Plates
A coating mixture comprising 12 μg/ml vitronectin and 12 μg/ml collagen (both included in the kit) was prepared by diluting each with 1×PBS.
The coating mixture was then added to the wells to cover the well surfaces (5 wells×2 plates were prepared). Plates were incubated overnight at room temperature. The coating mixture was then removed and the wells were rinsed once with PBS. Plates were aspirated right before use.
Cell Growth
Placenta derived cells (plcll-3-1) or bone marrow derived cells (BM108) were plated (200,000 cells per well) in 1 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco). Placenta derived cells (4 wells×2 plates) or bone marrow derived cells (1 well×2 plates) were grown until 100% confluent (typically overnight) before initiating osteogenic differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 1 ml osteogenesis induction medium (differentiation day 1). Osteogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 14-17 days.
As a control, one of the two plates (for each of the cell types) was not incubated with osteogenesis differentiation medium but rather with the growth medium (described hereinabove).
On day 17, osteocytes were fixed and stained with Alizarin Red Solution as depicted in detail below.
Staining Protocol
Osteocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in iced cold 70% ethanol for 1 hour at room temperature. The alcohol was then carefully aspirated and the cells were rinsed twice with water (5-10 minutes each wash). The water was then aspirated and alizarin red solution (500-1000 μl) was added to the cells. Cells were incubated with alizarin red solution at room temperature for 30 minutes. Alizarin red was removed and the cells were washed 4 times with 1 ml water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Experimental Results
Osteocyte differentiation of placenta—or bone marrow—derived adherent cells in osteogenic induction medium resulted in differentiation of over 50% of the bone marrow cells, as demonstrated by positive alizarin red staining (FIG. 8B). On the contrary, none of the placental derived cells of the present invention showed any signs of osteogenic differentiation (see FIG. 8E and Table 4, below).







TABLE 4







Differentiation summary













BM 108 +



Plc



BM109
PLC-11-3-1
PLC-8-2-1
Plc-15-3-4-2
4-3-1





Osteocytes
+++
−
−
−
−


Adipocytes
+++
−
−
−
−









Example 5
Comparison of Osteocyte Differentiation of 2D Adherent Cells of the Present Invention and Bone Marrow Cells in Modified Growth Medium
The adherent cells of the present invention (from placenta origin, at their 2D adherent cell stage) or bone marrow derived cells were grown under osteocyte differentiation stimulating conditions in a modified osteogenic medium comprising Vitamin D and higher concentrations of dexamethasone.
Materials and Experimental Methods
Osteogenesis Induction Medium
Osteogenesis induction medium was freshly made prior to each medium exchange using the components listed in Table 5, below, along with Vitamin D.







TABLE 5







Osteogenesis medium components










Component
Stock con
Amount
Final con














DMEM high glucose (Biological

8.7
ml
87%


Industries, Bet Haemek, Israel)






L-glutamine
×100
100
μl
×1


Serum (heat inactivated)

1
ml
10%


Dexamethasone (Chemicon)
10 mM
10
μl
10 μM


Asorbic Acid-2-Phosphate solution
0.1M
20
μl
0.2 mM 


(Chemicon)






Glycerol-2-Phosphate Solution
  1M
100
μL
10 Mm


(Chemicon)






Vitamin D (Sigma)
10 μM
10
μL
10 nM


Gentamycin (Biological Industries,
×100
100
μl
×1


Bet Haemek, Israel)









A 50 ml serum vial was heat inactivated, divided into 5 ml aliquots and kept at −20° C. until use.
Coating 48-well Tissue Culture Plates
A coating mixture comprising 12 μg/ml vitronectin and 12 μg/ml collagen (both from Chemicon) was prepared by diluting each with 1×PBS.
The coating mixture was then added to the wells to cover the well surfaces (5 wells×2 plates were prepared). Plates were incubated overnight at room temperature. The coating mixture was then removed and the wells were rinsed once with PBS. Plates were aspirated right before use.
Cell Growth
Placenta derived cells (PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1 fetal cells) were plated (100,000 cells per well) in 0.5 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) (4 wells×2 plates). Bone marrow derived cells (BM109) were plated (150,000 cells per well) in 0.5 ml growth medium (as described above) (1 well×2 plates). Cells were grown until 100% confluent (typically overnight) before initiating osteogenic differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 0.5 ml osteogenesis induction medium (differentiation day 1). Osteogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 26 days.
As a control, one of the two plates (for each of the cell types) was not incubated with osteogenesis differentiation medium but rather with the growth medium (described hereinabove).
On day 26, osteocytes were fixed and stained with Alizarin Red Solution as depicted in detail below.
Staining Protocol
Osteocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in iced cold 70% ethanol for 1 hour at room temperature. The alcohol was then carefully aspirated and the cells were rinsed twice with water (5-10 minutes each wash). The water was then aspirated and alizarin red solution (500-1000 μl) was added to the cells. Cells were incubated with alizarin red solution at room temperature for 30 minutes. Alizarin red was removed and the cells were washed 4 times with 1 ml water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Experimental Results
Osteogenic differentiation of placenta—or bone marrow—derived adherent cells was performed by modification of the protocol described in Example 4, hereinabove, according to previous teachings [Parloni et al. (2008) Stem Cells 26(2): 300-11]. The main difference between the growth conditions presented in Example 4 and the results presented herein was the addition of vitamin D to the differentiation medium and the higher concentrations of dexamethasone. As evident from the results, over 50% of the bone marrow cells underwent differentiation into osteocytes, as demonstrated by positive alizarin red staining (see FIG. 9B). However, none of the placental derived cells of the present invention showed any signs of osteogenic differentiation (see FIG. 9E and Table 4, hereinabove).
Example 6
Comparison of Adipocyte Differentiation of 2D Adherent Cells of the Present Invention and Bone Marrow Cells
The new adherent cells of the present invention (from placenta origin) were grown under adipocyte differentiation stimulating conditions at their 2D adherent cell stage in comparison to bone marrow derived cells.
Materials and Experimental Methods
Adipogenesis
Adipogenesis was carried out according to Chemicon adipogenesis kit (Chemicon adipogenesis kit, cat no. scr020, Millipore, Mass., USA)
Adipogenesis Induction Medium
Adipogenesis induction or maintenance mediums were freshly prepared prior to every medium exchange using the components depicted in Tables 6 and 7, below.







TABLE 6







Adipogenesis induction medium components










Component
Stock con
Amount
Final con














DMEM low glucose (Biological

4.4
ml
90%


Industries, Bet Haemek, Israel)






Serum (heat inactivated)

0.5
ml
10%












Dexamethasone (Sigma)
10 mM
0.5
μl
1
μM


IBMX (Sigma)
0.5M
5
μl
0.5
mM


Insulin (Sigma)
10 mg/ml
5
μL
10
μg/ml


Indomethacin (Sigma)
10 mM
50
μl
100
μM











Pen & Strep
×100
50
μl
×1
















TABLE 7







Adipogenesis maintenance medium components












Component
Stock con
Amount
Final con
















DMEM low glucose

4.4
ml
90%



Serum (heat inactivated)

0.5
ml
10%



Insulin
10 mg/ml
5
μL
10 μg/ml



Pen & Strep
×100
50
μl
×1









Cell Growth
Placenta derived cells (plc11-3-1) or bone marrow derived cells (BM108) were plated (200,000 cells per well) in 1 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco). Placenta derived cells (4 wells×2 plates) or bone marrow derived cells (1 well×2 plates) were grown until 100% confluent (typically overnight) before initiating adipogenesis differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 1 ml adipogenesis induction medium (differentiation day 1). Adipogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 25 days (as depicted in detail in Table 8, hereinbelow). Of note, monolayers of adipogenic cells were extremely fragile and could easily dislodged from plates, therefore, medium changes were performed with gentle medium changes to avoid disruption of the lipid droplets.
As a control, one of the two plates (for each of the cell types) was not incubated with adipogenesis differentiation medium but rather with the growth medium (described hereinabove).







TABLE 8







Adipogenesis differentiation schedule








Day
Medium











1
Adipogenesis Induction medium


3
Adipogenesis Induction medium


5
Adipogenesis Induction medium


7
Adipogenesis Maintenance medium


9
Adipogenesis Induction medium


11
Adipogenesis Induction medium


13
Adipogenesis Induction medium


15
Adipogenesis Maintenance medium


17
Adipogenesis Induction medium


19
Adipogenesis Induction medium


21
Adipogenesis Induction medium









On day 25, adipocytes were fixed and stained with oil red solution as depicted in detail below.
Staining Protocol
Adipocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in 4% Para formaldehyde for 30-40 minutes at room temperature. The fixative was then carefully aspirated and the cells were rinsed three times with PBS (5-10 minutes each wash). Next, the PBS was aspirated and the cells were rinsed twice in water. The water was then aspirated and oil red solution (500-1000 μl) was added to the cells. Cells were incubated with oil red solution at room temperature for 50 minutes. Oil red solution was removed and the cells were washed 4 times with 1 ml water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Preparation of Oil Red Solution
Stock of 0.25 g oil red (Sigma) was used which was dissolved in 50 ml iso-propanol by incubating 10-15 min in 37° C. bath.
For use, 30 ml of the stock stain was mixed with 20 ml DDW (left to stand for 10 minutes and then filtered with coffee filter paper). The oil red solution was prepared fresh for each use.
Experimental Results
Adipocyte differentiation of placenta—or bone marrow—derived adherent cells in adipocyte induction medium resulted in differentiation of over 50% of the bone marrow derived cells (see FIG. 8C), as demonstrated by positive oil red staining and by typical morphological changes (e.g. accumulation of oil droplets in the cytoplasm). In contrast, none of the placental derived cells of the present invention differentiated into adipocytes (see FIG. 8F and Table 4, hereinabove).
Example 7
Comparison of Adipocyte Differentiation of 2D Adherent Cells of the Present Invention and Bone Marrow Cells in Modified Growth Medium
The adherent cells of the present invention (from placenta origin, at their 2D adherent cell stage) or bone marrow cells were stimulated to differentiate into adipocytes in a modified adipocyte medium comprising a higher level of Indomethacine.
Materials and Experimental Methods
Adipogenesis Induction Medium
Adipogenesis induction medium was freshly prepared prior to every medium exchange using the components depicted in Table 9, below.







TABLE 9







Adipogenesis induction medium components










Component
Stock con
Amount
Final con














DMEM low glucose

4.4
ml
90%


Serum (heat inactivated)

0.5
ml
10%













Dexamethasone (Sigma)
1
mM
5
μl
1
μM












IBMX (Sigma)
0.5M
5
μl
0.5
mM













Insulin (Sigma)
10
mg/ml
5
μL
10
μg/ml


Indomethacin (Sigma)
10
mM
200
μl
100
μM


Gentamycine (Biological


10
μl




Industries)









Cell Growth
Placenta derived cells (PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1 fetal cells) were plated (100,000 cells per well) in 0.5 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) (5 wells×2 plates).
Bone marrow derived cells (BM109) were plated (100,000 cells per well) in 0.5 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) (4 well×2 plates). Cells were grown until 100% confluent (typically overnight) before initiating adipogenesis differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 0.5 ml adipogenesis induction medium (differentiation day 1). Adipogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 3-4 weeks.
As a control, one of the two plates (for each of the cell types) was not incubated with adipogenesis differentiation medium but rather with the growth medium (described hereinabove).
On day 26, adipocytes were fixed and stained with oil red solution as depicted in detail below.
Staining Protocol
Adipocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in 4% Para formaldehyde for 30-40 minutes at room temperature. The fixative was then carefully aspirated and the cells were rinsed three times with PBS (5-10 minutes each wash). Next, the PBS was aspirated and the cells were rinsed twice in water. The water was then aspirated and oil red solution (500-1000 μl) was added to the cells. Cells were incubated with oil red solution at room temperature for 50 minutes. Oil red solution was removed and the cells were washed 3 times with 1 ml double distilled water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Preparation of Oil Red Solution
Stock of 0.25 g oil red (Sigma) was used which was dissolved in 50 ml iso-propanol by incubating 10-15 min in 37° C. bath.
For use, 30 ml of the stock stain was mixed with 20 ml DDW (left to stand for 10 minutes and then filtered with coffee filter paper). The oil red solution was prepared fresh for each use.
Experimental Results
Adipocyte differentiation of placenta—or bone marrow—derived adherent cells was performed by modification of the protocol in Example 6, hereinabove, according to previous teachings [Parloni et al. (2007), supra]. The main difference between the growth conditions presented in Example 6 and the results presented herein was the higher concentration of Indomethacine. As evident from the results, over 50% of the bone marrow derived cells underwent differentiation into adipocytes (see FIG. 9C), as demonstrated by positive oil red staining and by typical morphological changes (e.g.
accumulation of oil droplets in the cytoplasm). In contrast, none of the placental derived cells of the present invention exhibited morphological changes typical of adipocytes (see FIG. 9F and Table 4, hereinabove).
Example 8
Biodistribution of PLX-C
Materials and Experimental Methods
Transfection of PLX-C Cells with Luciferase Expression Vector
PLX-C cells were stably infected with a lentiviral construct expressing the luciferase gene under the CMV promoter (FIG. 10).
Production of Infecting Virus
293TN producer cells were grown in DMEM medium (Gibco) supplemented with serum and antibiotics for 2-3 days (50-70% confluency) prior to transfection. A mixture of 10 μg of the packaging plasmid and 2 μg of expression construct and 20 of PlusTM Reagent (Invitrogen) were added to 400 μl of DMEM without supplements. The mixture was incubated for 15 min at room temperature (RT) and LipofectamineTM (30 μl dilutes in 400 μl of DMEM were added). The mixture was incubated at RT for 15 min. 293TN cells were washed and transferred to 2% serum media and transfection mixture was added. Cells were incubated in CO2 incubator at 37° C. over night and medium was collected 24-60 hrs post infection. Peak virus production was achieved after 48 hrs. Medium was collected, and centrifuged at 3000 rpm at room temperature for 5 minutes to pellet cell debris. Following centrifugation, the supernatant was filtered through Millex-HV 0.45 μm PVDF filters (Millipore, Cat. #SLHVR25LS).
Infection of PLX-C
PLX-C cells were seeded in a 24-well plate at a density of 0.6-1×105 cells per well in complete medium 24 hours prior to viral infection. After 24 hrs, 0.5 ml of virus suspension (diluted in complete medium with Polybrene at a final concentration of 5-8 μg/ml) was added. Cells were incubated for 24 hrs, then medium was replaced by complete DMEM medium and cells were incubated at 37° C. with 5% CO2 overnight. At day 4, the culture reached confluency and was split by 1:3 to 1:5, cells were allowed to grow for 48 hours in complete DMEM then cells were analyzed for Luciferase expression.
Efficiency rates of infection were close to 100%. Evaluation of luminescence in living cells and in living mice was performed using the IVIS Lumina Imaging system, which included a highly sensitive CCD camera that captured the luciferase luminescence signal.
Two weeks post infection 2×106 cells were injected IM or IV into SCID/Beige, NOD/SCID, SCID and Balb/C mice. Injected cells were monitored using the described IVIS system.
Experimental Results
As evident from the results, PLX-C cells continued to divide following infection, and expression levels of Luciferase in the growing cells remained strong and stable (FIG. 11).
Once PLX-C cells were injected into Balb/C mice, the biodistribution pattern was examined. As evident from the results, cells disappeared 72 hrs post IM injection (data not shown). However, PLX-C cells retained constant high levels of Luciferase expression, in vitro, for over three weeks (data not shown).
As shown in FIGS. 12A-D, cells injected IM into SCID/Beige mice immunodeficient mice retained up to 5 days at the site of injection and were not observed thereafter. PLX-C cells injected IV into SCID/Beige mice migrated after 24 hrs to the lungs, then to the site of injection (presumably homing to site of injury). Afterwards cells disappear gradually and were not observed after 3-4 weeks. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

## Claims

1. A bioreactor comprising:
a perfusion chamber housing:
i) a population of adherent cells from placenta that are negative for the marker CD200, wherein the adherent cells from placenta do not exhibit osteogenic differentiation when subjected to conditions that induce bone marrow cells to undergo osteogenic differentiation, wherein the adherent cells from placenta are present in the perfusion chamber under culturing conditions which allow for cell expansion, said conditions comprising perfusion of a cell culture medium through the bioreactor, and wherein the bioreactor is configured to adjust the rate of said perfusion according to the glucose concentration of the culture medium, and
ii) a plurality of 3D porrosive carriers on which the cells are cultured within the bioreactor, wherein each porrosive carrier comprises a fiber matrix, and wherein the carriers are packed in the perfusion chamber.

2. The bioreactor of claim 1, wherein more than 25% of the adherent cells from placenta are in S and G2/M cell cycle phases.

3. The bioreactor of claim 1, wherein the adherent cells from placenta are less committed to differentiation into adipogenic lineages as compared to adherent cells from bone marrow grown under the same conditions.

4. The bioreactor of claim 1, wherein the fiber matrix is composed of a non-woven fabric matrix.

5. The bioreactor of claim 4, wherein the non-woven fabric matrix comprises a polyester.

6. A bioreactor comprising:
a perfusion chamber configured for housing a population of adherent cells from placenta that are negative for the marker CD200 under culturing conditions which allow for cell expansion, said conditions comprising perfusion of a cell culture medium through the bioreactor, wherein the bioreactor is configured to adjust the rate of said perfusion according to the glucose concentration of the culture medium, and wherein the adherent cells from placenta do not exhibit osteogenic differentiation when subjected to conditions that induce bone marrow cells to undergo osteogenic differentiation, wherein the adherent cells from placenta are present in the perfusion chamber.