Source: http://www.google.com/patents/US8137965?dq=7565338
Timestamp: 2017-08-20 07:18:55
Document Index: 714011028

Matched Legal Cases: ['§371', 'Application No. 60', 'application No. 10012560', 'Application No. 2003273292', 'Application No. 2003273292', 'Application No. 2009240818', 'Application No. 2', 'Application No. 03755791', 'Application No. 12', 'Application No. 2003', 'Application No. 03755791']

Patent US8137965 - Methods and compositions for tissue regeneration - Google Patents
A kit is disclosed that includes a first component comprising alginate, wherein the first component is comprised in a first sterile vial, and a second component comprising cells comprising keratinocytes or fibroblasts, or mixtures thereof, that secrete one or more biologically active molecules selected...http://www.google.com/patents/US8137965?utm_source=gb-gplus-sharePatent US8137965 - Methods and compositions for tissue regeneration
Publication number US8137965 B2
Application number US 12/967,619
Also published as CA2497193A1, CA2497193C, CA2904030A1, EP1545565A1, EP1545565A4, EP1545565B1, EP2311469A2, EP2311469A3, US7144729, US7449333, US7700351, US7879605, US8323638, US8679475, US9173906, US20030165482, US20060121002, US20090232791, US20100068281, US20110083987, US20120134962, US20130052168, US20140377229, WO2004022077A1
Publication number 12967619, 967619, US 8137965 B2, US 8137965B2, US-B2-8137965, US8137965 B2, US8137965B2
Inventors Eric Rolland, Thomas Hunziker, Beatrice Mis, Christopher Rinsch
Original Assignee Dfb Technology Holdings, Llc
Patent Citations (44), Non-Patent Citations (38), Referenced by (1), Classifications (63), Legal Events (5)
US 8137965 B2
14. The kit of claim 13, wherein the first and second pouches are fabricated of a material capable of withstanding temperatures ranging from −80° C. to −160° C.
This application is a continuation of co-pending U.S. application Ser. No. 12/562,767, filed Sep. 18, 2009, which is a continuation of U.S. application Ser. No. 12/255,481, filed Oct. 21, 2008 (now issued as U.S. Pat. No. 7,700,351), which is a continuation of U.S. patent application Ser. No. 10/526,853 filed Jan. 9, 2006 (now issued as U.S. Pat. No. 7,449,333), which is a U.S. national phase application under 35 U.S.C. §371 of PCT Application No. PCT/US2003/027888 filed on Sep. 5, 2003, which claims priority to U.S. patent application Ser. No. 10/324,257 filed on Dec. 19, 2002 (now issued as U.S. Pat. No. 7,144,729), which claims the benefit of U.S. Provisional Application No. 60/408,565 filed on Sep. 6, 2002. The contents of all of the above-referenced applications are incorporated by reference.
The cell types may naturally secrete the one or more biologically active molecules or they may be genetically engineered to secrete an exogenous level of the one or more biologically active molecules. Secretion may be controlled by gene switching or it may be constitutive. In one embodiment, the first component contains fibrinogen. In another embodiment, the first component contains fibrinogen and the second component contains from about 1×103 cells/μl to about 50×103 cells/μl The second component also contains thrombin and can optionally contain a cryoprotectant such as a 10% glycerol solution, a 15% glycerol solution, and a 15% glycerol and 5% human serum albumin solution.
The number of cells landing on the target area of the patient (i.e., the number of cells per cm2 of patient) will vary depending on the concentration of each of the components and the ratios of keratinocytes to fibroblasts used in component #2. Those skilled in the art will recognize that the concentration of cell in the second component of the cell preparation can be varied from about 1×103 cells/μl to about 50×103 cells/μl For example, in some embodiments, the number of cells/0 of the cell preparation component #2 can range from about 5×103 cells/μl to about 20×103 cells/μl. Thus, if two sprays of approximately 130 μl/spray are administered to a patient, approximately about 1.3×106 to about 5.2×106 cells are administered to the patient.
PROPERTY UNITS Value (typical)
Water Vapor Transmission Rate g/m2 · d @ 38° C., 90% R.H. <0.5
Oxygen (O2) Transfer Rate cc/m2 · d · atm@ 23° C., 50% R.H. <2.0
The sealing parameters for a suitable pouch will depend on the particular sealing equipment used and its compatibility with the sealant layer of the secondary substrate. However, typical parameters for sealing can include: temperature=100−140° C.; dwell time=0.30−0.75 sec; and pressure=60−80 psi.
In another embodiment, the vials may be sealed in a rigid, transparent container, fabricated by using a polymer resistant to temperatures below −70° C. to as low as −196° C. A variety of different types of vials may be used for freezing components #1 and #2. In one preferred embodiment, a novel vial (5) such as that shown in FIG. 6 a and FIG. 6 b is employed. This vial is used to freeze down the components of the cell preparation of the invention. Upon thawing, the screw-on cap (4) closing the vial can be replaced with a spray pump applicator. In one embodiment, bottle may be made of polypropylene, which is resistant to the low temperatures employed in the cryopreservation protocol. The wall thickness of this vial (1) should be approximately 0.8 mm to facilitate heat/cold transfer across the wall, which is important for both the freezing and thawing processes. Additionally, the vial can be designed to stand upright after a spray pump has been screwed on, and the bottom of the vial (2) is conical (3) to facilitate emptying of the contents.
Example 1 Isolation of Keratinocytes and Fibroblasts
Example 2 Composition of the Components of the Cell Preparation of the Invention
HESS with Ca2+ and Mg2+ was the chosen diluent.
Example 3 Testing the Effective Dose of Mitomycin C
The rat fibroblast cell line (CRL 1213), the FGF1-transfected rat fibroblast cell line (1175/CRL 1213), the human telomerase immortalized fibroblast line (MDX12), and the primary human fibroblasts (EDX1) were each growth arrested using the following method. Fibroblasts were grown in DMEM+10% FCS, 25 mM Hepes, 1 mM pyruvate, 2 mM L-Gln, 100 U/ml penicillin, 100 μg/ml streptomycin in T75 flasks. At confluency, the cells were detached and plated at a density of 105 cells/cm2, further incubated for 48 h, then treated with mitomycin C (MMC) at 0, 2, 4, 8, 12 μg/ml for 5 h. The cells were then rinsed with PBS and detached with 0.05% trypsin/0.02% EDTA. The remaining cells were plated at densities of 100 to 5000 cells/cm2 in T25-flasks respectively (10 flasks for each density). These cells were incubated at 37° C. with 2 media changes per week.
Example 4 Testing Effective Dose of Gamma Irradiation on Fibroblasts and Keratinocytes
Example 5 Testing Cell Densities with Fibrin Paste: Secretion of Growth Factors and Cytokines by Mixtures of Keratinocytes and Fibroblasts in a Fibrin Matrix
Cell GM-CSF (pg) VEGF (pg) KGF (pg) HGF (pg) IL-1 beta (pg)
2.5 million/ml 9 5 1002 80 90 6 1869 144 29 4
Example 6 Allox Phase I Clinical Trial C2001
Component #1 A liquid suspension of fibrinogen; (1 spray=50 μl)
Component #2 A liquid suspension of keratinocytes and fibroblasts (Ratio 1:1, 15×106 cells/ml) mixed with thrombin; (1 spray=50 μl)
Ulcer Ulcer Ulcer surface % of area
surface surface area (cm2) at % of area reduction
area (cm2) area (cm2) 4 Weeks reduction SD1-
Patient ID Center at SD1 at SD57 Follow Up SD1-SD57 Foltow Up
200142 4 20.0 14.0 10.3 30 43
Example 7 Testing Optimal Dilutions of Tisseel VII (Fibrin Glue) and Standard Human Plasma
Fibrin is one potential biological polymer that can be employed for suspending and trapping cell mixtures for therapeutic purposes. Polymerized fibrin is created by mixing fibrinogen and thrombin together at appropriate concentrations. In the matrix shown in FIG. 3, various dilutions of the fibrinogen (Tisseel, Baxter) and thrombin (Tisseel, Baxter) were tested for their effect on the polymerization process and generation of the end product fibrin. Dilutions considered ranged from ¼ to 1/80 of the original fibrinogen and thrombin components supplied in the TissuCol kit. In the original Tisseel kit, fibrinogen had a concentration of 75 to 115 mg/ml, while thrombin had a concentration of 500 IU/ml. For this study, the fibrinogen was diluted in HBSS without Ca2+ and Mg2+ and the thrombin was diluted in HBSS with Ca2+ and Mg2+.
Example 8 Comparison of Growth Factor Release From Sprayed Versus Non-Sprayed Cells
Example 9 Comparison of Growth Factor Production By Different Keratinocyte:Fibroblast Cell Ratios
In FIG. 5 a and FIG. 5 b, a comparison is made of growth factors released from cells entrapped in a fibrin matrix. The fibrin cell matrix is formed by spraying either one or two sprays of component #2 (cells+thrombin) with one or two sprays of component #1 (fibrinogen). A concentration of 15×106 cells/ml in component #2 was employed and the spray pump used delivered a volume of 70 μl per spray. To growth arrest cells, both primary human fibroblasts and keratinocytes were treated with 8 ug/ml mitomycin during 5 hours. Cells were rinsed with HBSS without Ca2+ and Mg2+ prior to trypsinization.
In FIG. 5 a, one spray (1.05×106 cells)=1 spray of cells+thrombin (70 μl) and 1 spray of fibrinogen (70 μl). In FIG. 7 b, two sprays (2.1×106 cells)=2 spray of cells+thrombin (140 μl) and 2 sprays of fibrinogen (140 μl).
Example 10 Comparison of Storage at −160° C. Versus −80° C. During One Week
FIG. 7 shows a comparison of growth factor secretion by cells stored cryopreserved at −160° C. versus at −80° C. for a period of one week. The cryoprotectant used in this experiment was a 10% glycerol solution with 10% thrombin (Tisseel, Baxter) and the keratinocyte:fibroblast ratio employed was 1:1. Prior to cryopreservation, cells were detached from their culture surfaces using trypsin and subsequently irradiated using gamma (γ) irradiation at 80 Gy. A controlled rate freezer was used to gradually cool cell preparations to −80° C. After thawing one week later, one spray (130 μl) of the cell preparation (1.3 million cells at 10 million cells/ml) were spray mixed with one spray (130 μl) of fibrinogen in single wells of a 24 well petri dish. The results show GM-CSF, VEGF, and bFGF secretion during day 2 for three samples stored for one week at −160° C. and four samples stored for one week at −80° C. compared to a control fresh (unfrozen) sample containing the same cryoprotectant. Secreted GM-CSF and VEGF were dosed in the culture media, while bFGF was dosed in the fibrin matrix. Secretion data indicates that both −80° C. and −160° C. are suitable for storage, though −160° C. may be preferable when using a 10% glycerol solution. Data is shown as average±SEM (n=4).
Example 11 Comparison of Secretion After One Week Storage at −80° C. in 10% Glycerol Versus 15% Glycerol.
FIG. 8 shows a comparison of GM-CSF, VEGF and bFGF secretion by cells cryopreserved at −80° C. in 10% glycerol versus in 15% glycerol for a period of one week. The keratinocyte:fibroblast ratio used in this example was 1:1 mixed with 10% thrombin (Tisseel, Baxter) and a cryoprotectant. Prior to cryopreservation, cells were detached from their culture surfaces using trypsin and subsequently irradiated using gamma (γ) irradiation at 80 Gy. A controlled rate freezer was used to gradually cool cell preparations to −80° C. After thawing one week later, one spray (130 μl) of the cell preparation (1.3 million cells at 10 million cells/ml) were spray mixed with one spray (130 μl) of fibrinogen in single wells of a 24 well petri dish.
Example 12 Comparison of Secretion After One Week Storage at −80° C. in 15% Glycerol Versus 15% Glycerol+5% HSA
FIG. 9 shows a comparison of growth factor secretion by cells cryopreserved at −80° C. in 15% glycerol versus 15% glycerol+5% Human Serum Albumin (HSA) (Griffols) for a period of one week. The keratinocyte:fibroblast ratio used in this example was 1:3 mixed with 10% thrombin (Tisseel, Baxter) and a cryoprotectant. Prior to cryopreservation, cells were detached from their culture surfaces using trypsin and subsequently irradiated using gamma (γ) irradiation at 80 Gy. A controlled rate freezer was used to gradually cool cell preparations to −80° C. After thawing one week later, one spray (130 μl) of the cell preparation (1.3 million cells at 10 million cells/ml) were spray mixed with one spray (130 μl) of fibrinogen in single wells of a 24 well petri dish. The results show GM-CSF, VEGF, and bFGF secretion during day 2 for three samples stored for one week at −80° C. using 15% glycerol and three samples stored for one week at −80° C. using 15% glycerol+5% HSA (Griffols) compared to control fresh (unfrozen) samples containing the same cryoprotectants. Secretion data indicates that the addition of Human Serum Albumin improves the frozen product formulation by permitting higher protein secretion levels. Data is presented as average±SEM (n=4).
Example 13 Bioactivity of Keratinocyte and Fibroblast Mixtures Following Long-Term Storage at −80° C.
FIG. 10 details the secretion of the human proteins GM-CSF and VEGF by cell preparations following storage at a temperature of −80° C. for extended periods. Data from three separate clinical production batches is shown. Batches containing a ratio of human primary fibroblasts to keratinocytes of 1:1 at a final concentration of 10×106 cells/ml were irradiated using gamma (γ) irradiation at 80 Gy and frozen in a solution containing thrombin, 15% glycerol and 5% human serum albumin. Samples from each production batch were thawed following 1, 4, 8, and 12 weeks storage at −80° C. Thawed samples were subsequently sprayed into 24-well plates for testing. In individual wells, a single spray (130 μl) of cells+thrombin+cryoprotectant is mixed with a single spray (130 μl) of fibrinogen. The mixture of these two sprays creates a fibrin polymer matrix containing living fibroblasts and keratinocytes. The secretion of proteins by cells trapped in the fibrin matrix is measured during day 2 (the period lasting from 24 hours to 48 hours after thawing).
Example 14 Comparison of Secretion After One Week Storage at −80° C. for Different Keratinocyte:Fibroblast Ratios
FIG. 11 shows the secretion of growth factors from cryopreserved cell preparation formulations following one week of storage at −80° C. The graph shows differences in secretion for various human primary keratinocyte:fibroblast ratios, including 1:0, 1:1, and 1:9 as well as differences associated with total cell concentrations of 5, 10 and 20 million cells/ml. Prior to cryopreservation, cells were detached from their culture surfaces using trypsin and subsequently irradiated using gamma (γ) irradiation at 80 Gy. A controlled rate freezer was used to gradually cool cell preparations to −80° C. After thawing one week later, 25, one spray (130 μl) of the cell preparation (5, 10 and 20 million cells/ml) were spray mixed with one spray (130 μl) of fibrinogen in single wells of a 24 well petri dish.
Example 15 Comparison of Growth Factor Secretion in Fresh and Cryopreserved Samples Stored at −80° C.
Prior to cryopreservation, cells were detached from their culture surfaces using trypsin and subsequently irradiated using gamma (γ) irradiation at 80 Gy. Cell concentrations tested included 5, 10, and 20 million cells/ml (with a keratinocyte:fibroblast ratio 1:1), each mixed with 10% thrombin (Tisseel, Baxter) and the cryoprotectant. A controlled rate freezer was used to gradually cool cell preparations to −80° C. After thawing one week later, one spray (130 μl) of the cell preparation (5, 10 and 20 million cells/ml) were spray mixed with one spray (130 μl) of fibrinogen in single wells of a 24 well petri dish. To assess variability in frozen (cryopreserved) samples, 3 separate tubes for each condition were cryopreserved for one week at −80° C. Upon thawing, five samples were made per tube. The data presented in Table 8 is the average of the 15 samples available for each condition. The reproducible secretion data observed in the three frozen and thawed tubes per condition attests to the quality of the cryopreservation method. For fresh preparations, either 3 or 4 samples were made. Secretion of GM-CSF and VEGF by both freshly trypsinized and frozen cell preparations was observed to increase as cell density in the fibrin matrix increased from 2.5 to 5 to 10 million cells/ml (corresponding to 5, 10 and 20 million cells/ml found in original frozen preparations). This further illustrates the potential of dosing therapeutic effects by cell-based treatments.
Sample Sample % %
Name No. Average SEM Fresh Average SEM Fresh
5 million/ 4 40 14 100 1433 289 100
10 million/ 4 270 128 100 3418 1293 100
20 million/ 3 751 16 100 5628 1150 100
5 million/ 15 81 11 202.5 986 96 68.8
ml, 1 week
at −80° C.
10 million/ 15 254 21 94.1 2135 254 62.5
20 million/ 15 554 51 73.8 3462 335 61.5
ml,1 week
Example 16 Evaluation of the Safety of the Frozen Cell Preparation of the Invention
No serious adverse events were reported in relation to this cell preparation. Four moderate to severe adverse events were thought to be attributable to the cell preparation (3× ulcer pain, 1× with increasing ulcer size). Moreover, there were no clinical signs of wound infection. Complete closure at week 12 was observed in 10 patients, 7 within 4 weeks and 3 within 4 to 12 weeks of treatment. Mean time to closure was 5.4 weeks.
Example 17 Spray Applied Living Keratinocytes and Fibroblasts as a Biologically Active Wound Dressing
US5261255 Nov 8, 1991 Nov 16, 1993 Instacool Inc. Of North America Device for fractionating constituent components of a substance using cryoprecipitation
US5902608 Dec 31, 1996 May 11, 1999 University Of North Carolina At Chapel Hill Surgical aids containing pharmaceutically acceptable fixed-dried human blood platelets
US6010887 Sep 14, 1994 Jan 4, 2000 Imperial Chemical Industries Plc Regulation of gene expression
US6479052 Dec 2, 1999 Nov 12, 2002 Bristol-Myers Squibb Company Spray delivery of cells
US6548297 Nov 9, 1995 Apr 15, 2003 Celadon Science, Inc. Sheets of cultured epithelial cells used for wound repair and their cryopreservation
US6720009 May 28, 2002 Apr 13, 2004 Biora Bioex Ab Matrix protein compositions for treating infection
US7144729 * Dec 19, 2002 Dec 5, 2006 Dfb Pharmaceuticals, Inc. Methods and compositions for tissue regeneration
US7449333 * Sep 5, 2003 Nov 11, 2008 Dfb Pharmaceuticals, Inc. Methods and compositions for tissue regeneration
US7700351 * Oct 21, 2008 Apr 20, 2010 Dfb Pharmaceuticals, Inc. Methods and compositions for tissue regeneration
US20020048563 Aug 30, 2001 Apr 25, 2002 Baetge E. Edward Methods and compositions for tissue regeneration
DE19949290A1 Oct 12, 1999 Apr 26, 2001 Albrecht Bettermann Partikuläres Konstrukt zur Verwendung in der Transplantationsmedizin
ES2132027A1 Title not available
JP2000501299A Title not available
JP2001517431A Title not available
WO1997006835A1 Aug 12, 1996 Feb 27, 1997 Smith & Nephew Plc Cell culture products
WO1999015637A1 Sep 15, 1998 Apr 1, 1999 V.I. Technologies, Inc. Fibrin microbeads and uses thereof
WO2000025838A1 Nov 4, 1999 May 11, 2000 Baxter International Inc. Element provided with a fibrin layer, preparation and use thereof
WO2000032207A1 Dec 2, 1999 Jun 8, 2000 Bristol-Myers Squibb Company Spray delivery of cells
WO2001003750A1 Jul 6, 2000 Jan 18, 2001 Advanced Tissue Sciences, Inc. Human naturally secreted extracellular matrix-coated device
WO2002072800A1 Feb 28, 2002 Sep 19, 2002 Centro De Investigaciones Energeticas Medioambientales Y Tecnologicas (C.I.E.M.A.T.) Artificial dermis and production method therefor
WO2002078721A1 Apr 2, 2002 Oct 10, 2002 Bio Tissue Technologies Ag Two-constituent compositions for the in situ production of cell transplants that comprise fibroblasts and keratinocytes
1 Badiavas, et al., "Retrovirally Mediated Gene Transfer in a Skin Equivalent Model of Chronic Wounds", J. Dermatol. Sci., 13:56-62, 1996.
2 Cooper, et al., "Use of a Composite Skin Graft Composed of Cultured Human Keratinocytes and Fibroblasts and a Collagen-GAG Matrix to Cover Full-Thickness Wounds on Athynic Mice", Surgery, 109:198-207, 1991.
3 Currie, et al., "The Use of Fibrin Glue in Skin Grafts and Tissue-Engineered Skin Replacements: A Review", Plast. Reconstr. Surg., 108:1713-1726, 2001.
4 Database Prous Science Intergrity (Online), "Modex advances Alox through early-stage clinical testing," Database accession No. 323719, Aug. 7, 2002.
5 Database WPI, "Artificial skin production-comprises cell cultivation of keratinocytes on base of gel of human fibrin populated with human fibroblasts," Section Ch., Week 199940, Derwent Publications, Ltd., AN 1999-471189, 1999.
6 Davies, "Synthetic materials for covering burn wounds: progress towards perfection. Part I. Short term dressing materials," Burns Incl Therm Inj. 10:94-103, 1983.
7 Del Rio, I., "A Preclinical Model for the Analysis of Genetically Modified Human Skin In Vivo," Human Gene Therapy, 13:959-968, 2002.
8 Extended European Search Report, issued in European patent application No. 10012560.8, dated Mar. 9, 2011.
9 Falanga et al., "Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent," Archives of Dermatology, 134(3):293-300, 1998.
10 Hunt et al., In: Current Surgical Diagnosis & Treatment pp. 86-98, 1988.
11 Hunt et al., In:. The Surgical Wound, Dineen & Hildrick-Smith, eds., pp. 1-18, 1981.
12 Kannon and Garrett, "Moist wound healing with occlusive dressings. A clinical review," Dermatol. Surg., 21:583-590, 1995.
13 Kolodka et al., "Evidence for Keratinocyte Stem Cells in vitro: Long Tenn Engraftment and Persistence of Transgene Expression from Retrovirus-Transduced Kerationocytes," Proc. Nat!. Acad. Sci. USA. 95:4356-4361. 1998.
14 Kuroyanagi et al., "A Cultured Skin Substitute Composed of castsand Kerationocytes with a Collagen Matrix: Preliminary Results of Clinical Trials," Ann. Plait. Surg., 31:340-351, 1993.
15 Langdon et al., "Reconstitution of Structure and Cell Function in Human Skin Grafts Derived from Cryopreserved Allogeneic Dennis and Autologous Cultured Keratinocytes," J. Invest. Dermatol., 91:478-485 , 1988.
16 Maruguchi, et al., "A New Skin Equivalent: Kerationocytes Proliferated and Differentiated on Collagen Sponge Containing Fibroblasts," Plast. Reconstr. Surg., 93:537-546, 1994.
17 Office Communication, issued in Australian Patent Application No. 2003273292, dated Dec. 13, 2007.
18 Office Communication, issued in Australian Patent Application No. 2003273292, dated Jan. 30, 2009.
19 Office Communication, issued in Australian Patent Application No. 2009240818, mailed on Nov. 29, 2011.
20 Office Communication, issued in Canadian Patent Application No. 2,497,193, dated Dec. 31. 2010.
21 Office Communication, issued in European Patent Application No. 03755791.5, dated Jul. 9, 2008.
22 Office Communication, issued in European Patent Application No. 12/967,619, dated Jan. 10, 2011.
23 Office Communication, issued in Japanese Patent Application No. 2003-157362, dated Oct. 15, 2009. (English Translation).
24 Office Communication, issued in U.S. Appl. No. 10/324,527, dated Apr. 13, 2005.
25 Office Communication, issued in U.S. Appl. No. 10/324,527, dated Apr. 14, 2006.
26 Office Communication, issued in U.S. Appl. No. 10/324,527, dated Jul. 22, 2005.
27 Office Communication, issued in U.S. Appl. No. 10/526,853, dated Jan. 8, 2008.
28 Office Communication, issued in U.S. Appl. No. 10/526,853. dated Aug. 21, 2008.
29 Office Communication, issued in U.S. Appl. No. 12/255,481, dated Jun. 18, 2009,
30 Office Communication, issued in U.S. Appl. No. 12/562,767, dated Mar. 17, 2010.
31 PCT International Search Report issued in International Application No. PCT/US01/27104, dated Apr. 8, 2002.
32 PCT International Search Report issued in International Application No. PCT/US03/27888, dated Dec. 4, 2003.
33 Richey et al. "Topical growth factors and wound contraction in the rat: Part I. Literature review and definition of the rat model," Annals of Plastic Surgery 23(2): 159-165, 1989.
34 Riley, "Wound healing," Am. Pam. Physician 24: 107-113, 1981.
35 Singer et al., "Cutaneous Wound Healing," New England Journal of Medicine, 341:738-746, 1999.
36 Sugihara, et al., "Effects of Fat Cells on Keratinocytes and Fibroblasts in a Reconstructed Rat Skin Model Using Collagen Gel Matrix Culture," British J. Dennatol., 144:244-253, 2001.
37 Supplementary European Search Report issued in European Patent Application No. 03755791.5, dated Mar. 28, 2008.
38 Winter, "Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig," Nature, 193: 293.294, 1962.
WO2016154206A1 Mar 22, 2016 Sep 29, 2016 Smith & Nephew, Inc. Biopreservation medium and uses for biopreservation of biological materials
U.S. Classification 435/325, 435/366, 435/371
International Classification A61K38/18, A61F13/00, A61K35/36, C12N5/071, A61K38/19, A61K38/20, A61L31/00, C12N5/00, A61K35/12, C12N5/07, A61L15/44, C12N5/10, C12N5/22, A61K48/00, A61L27/00, A61L27/38, C12N5/077, A61L26/00, A61L15/40
Cooperative Classification C12N2510/04, C12N2510/00, C12N2502/1323, C12N2502/094, C12N5/0698, C12N5/0656, A61L27/3886, A61L27/3813, A61L27/3804, A61L27/3633, A61L27/3616, A61L26/0057, A61L15/40, A61K48/00, A61K47/10, A61K41/0023, A61K38/4833, A61K38/363, A61K38/19, A61K38/18, A61K9/19, A61K9/10, A61K9/0014, A61K35/33, A61K35/36
European Classification A61K35/12, A61K38/20, A61K35/36, A61K38/18, A61K38/19A, C12N5/06B13F, A61L27/36B14, A61L15/40, A61L27/38F, A61L26/00F, C12N5/06T2, A61K38/19B, A61L27/38B4, A61L27/38B, A61K38/48K5, A61K38/36A
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