Controlled hydrodynamic cell culture environment for three dimensional tissue growth

A novel hydrodynamic cell culture environment is disclosed for a two-chamber roller bottle. The unstable hydrodynamics of a gas headspace media chamber is coupled through a center opening to a second zero headspace culture chamber. Coupling the two chambers creates novel fluid streamlines that add turbulence for improved mass transfer and slow media repletion. The exchange between chambers regulates turbulence and thus concomitant exchange of nutrients i.e., environmental control. It is disposable, automatable, and suited to culturing tissue for transplants.

FIELD OF THE INVENTION 
The present invention relates a novel hydrodynamic cell culture environment 
embodied in a two chamber roller bottle with a controlled environment and 
uniquely combines; turbulence, wall shear, gas-to-liquid mass transfer, 
and cell culture environmental control, to the engineering of tissue 
constructs. Environmental parameters are aseptic and controlled, therein 
engineering the outcome of the tissue construct. The invention herein 
disclosed is especially suited to the culture of autologous tissues for 
clinical transplants, methods for engineering tissue constructs; and the 
product from the process. Tissue constructs derived from the novel 
hydrodynamic culture environment of this invention are phenotypically 
different from tissue constructs obtained by other culture vessels. 
BACKGROUND OF THE INVENTION 
The culture process for mammalian cells, animal cells, insect cells, 
bacteria, yeast and molds has one major rate-limiting step, oxygen mass 
transfer. Oxygen metabolism is essential for metabolic function. In 
mammalian and animal cell culture, oxygen flux is especially important 
during the early stages of rapid cell division. Oxygen utilization per 
cell is greatest when cells are suspended; requirements decrease as the 
cells aggregate and differentiate. However, during the later phases of 
cell culture, as the number of cells per unit volume increases, the bulk 
oxygen mass transfer requirements increase. Traditionally, increased 
requirements are accommodated by mechanical stirring methods. The present 
invention integrates several features needed for the engineering of 
autologous tissue transplants. Incorporated into the invention are; 
automated cell culture media repletion, the capability to grow cells in 
monolayer, a turbulent hydrodynamic cell culture environment suited to the 
growth of three-dimensional tissue constructs, a unique configuration that 
allows both monolayer and 3-dimensional culture in a variety of 
combinations, a gas head space for direct oxygenation of the cell culture 
media, an internal sensor for aseptically monitoring media changes, the 
capability to aseptically regulate both media repletion and add turbulence 
for improved mass transfer, and all features are included in a very low 
cost disposable two chamber tissue culture roller bottle. It will be 
appreciated by those of ordinary skill in the art, that the invention 
eliminates a large portion of the cell culture labor costs. The instant 
invention; 1) establishes near physiological control for improved 
engineering of tissue constructs, 2) enhances mass transfer through 
hydrodynamic shear forces generated at the wall and through the turbulent 
interface with the first chamber, 3) eliminates cellular contact with a 
potentially toxic silicone membrane oxygenator because oxygenation is 
predominately through the air fluid interface, 4) increases the 
concentration of de novo growth factors because the media is not replaced 
as often, and 5) greatly reduces the risk of contamination. 
Culturing tissue for transplantation requires that several conditions be 
met before the tissue receives Food and Drug Administration (FDA) 
approval. Those FDA conditions include; functionality that ameliorates the 
disease; consistent and reproducible growth of tissue constructs; and 
proven sterility. To achieve in vivo functionality, engineered tissue 
constructs must be three-dimensional. To be reproducible, the cell culture 
environment should be regulated to match human physiology, a feature of 
the invention. Data from the aseptic monitoring of the growing construct 
can be used to validate sterility and establish specifications. 
Transplantable tissue has three key features; 1) an extracellular matrix 
for mechanical stability and scaffolding; 2) cell-to-cell contact to 
maintain viability and function; and 3) a three-dimensional shape to 
segregate cell subpopulations for growth and proliferation. Standard 
tissue culture approaches (e.g.; t-flasks, petri dishes, microgravity 
culture vessels, roller bottles and stirred roller bottles) have 
consistently failed to yield transplantable tissue that directly supplants 
organ function. Failure is often related to the loss of multi-dimensional 
cell-to-cell contacts and the overgrowth of unwanted cell subpopulation. 
In roller bottles and stirred reactors, cells are prevented from sustained 
three-dimensional growth. Shear preselects for only those cell 
subpopulations that are robust to its damage. The present invention 
controls the addition of turbulence and concomitant shear, and localizes 
shear to specific areas. There are other selection pressures that impede 
normal growth, besides those intrinsic to standard cell culture roller 
bottles. Specifically, those technical operations associated with feeding 
the culture. Standard cell culture practice replaces cell culture media at 
regular intervals or when the media changes color. Replacing the media at 
regular intervals dilutes growth factors that are vital to cell growth and 
proliferation, and typically exposes cells to abnormally high oxygen 
concentrations. Abnormally high oxygen exposure occurs when spent cell 
media is replaced with fresh media. Fresh media equilibrated to room air 
has a dissolved oxygen concentration of approximately 159 mm Hg, often 
more than double the normal concentration found in the average mammalian 
interstitial space (normal 40-80 mm Hg). High levels of oxygen are known 
to be toxic to highly differentiated cells. Therefore, the practice of 
replacing conditioned media with fresh media equilibrated to ambient air, 
preselects for cells that are less sensitive to oxygen toxicity (e.g.; 
fibroblasts). Changing media based on the pH indicator dye, phenol red, 
imposes another common environmental selection pressure. Human physiology 
maintains the vascular and interstitial pH between 7.30-7.40. Deviation 
beyond physiological limits often results in mental confusion and organ 
dysfunction. Yet, most cell culture facilities routinely maintain the pH 
within 7.00 to 7.45. If patients were repeatedly exposed to standard cell 
culture pH extremes, they would eventually succumb to multi-system organ 
failure. 
There are several deficiencies associated with continuous perfusion of cell 
cultures. Continuous perfusion consumes large amounts of cell culture 
media, serum, and growth factor supplements. The expenses associated with 
perfusion reactors preclude their general use in research, and perfusion 
often yields tissue constructs that are delicate and of poor 
functionality. There are several additional drawbacks to profusion 
culture; cell populations are first manually expanded as monolayers of 
cells, yielding an atypical architecture; subpopulations that are robust 
and grow rapidly, overgrowing the other populations; and the cells must be 
anchorage dependant to keep from being washed away in the perfusion 
stream. 
A three-dimensional architecture is required for tissue constructs to be 
functional. As aggregates of cells organize, they exhibit the architecture 
of nascent tissue. To create a tissue construct, dissimilar cell types 
must aggregate, free from the selection pressures that force cells to 
spread and grow along a flat surface. Hydrogels and media viscosity 
enhancers permit cell mixtures to remain in contact with other cells, 
thereby allowing three-dimensional tissue growth. However, it has been 
observed that the sizes of the constructs were small, and latter deduced 
that they were mass transfer limited. 
Roller bottles are ubiquitous to cell culture facilities, and have been in 
use for decades. Their strengths are; they are disposable and offer a 
large surface for cell attachment. Their weakness is a consequence of the 
large uncontrolled hydrodynamic shear associated with a gas headspace and 
the abundance of turbulent eddies. The high shear environment inherent in 
roller bottles precludes their use for growing tissue for transplantation. 
Only those cell types that are not damaged by the shear and adhered to the 
wall, can be maintained in culture for the extended periods. 
Horizontal rotation for suspension i.e., clinostatic rotation, began in the 
1700s. The physics for growing cells in a free fall or in a microgravity 
like environment, was first characterized in detail by the Argonne 
National Laboratory (R. R. Dedolph and M. H. Dipert, 1971). Those 
principles were applied in the NASA microgravity culture vessels and cell 
culture methods, and the vessel and culture methods were first disclosed 
by M. Lewis et. al., May 1987. Later, the tubular silicone oxygenator in 
the culture vessel of M. Lewis et. al., 1987, was replaced by a membrane 
silicone oxygenator, and the culture vessel patented by NASA--U.S. Pat. 
No. 5,026,650 issued June 1991. Culture systems developed by NASA strive 
to achieve a quiescent microgravity like environment, having origins in 
the NASA space program. Microgravity culture conditions are created by low 
shear and essentially no relative motion of the culture environment with 
respect to the walls of the culture vessel (U.S. Pat. No. 5,496,722; claim 
12). Essentially no relative motion differences between the culture media 
and the vessel wall, is often referred to as solid body rotation. The 
forces maintaining solid body rotation are associated with the 1:1 viscous 
coupling between the vessel wall and the cell culture media contained in 
the vessel (G. F. Spaulding, 1994). However, microgravity is known to 
cause aberrancy. There are over 100 scientific articles and abstracts 
demonstrating or suggesting microgravity induced aberrancy in cell growth, 
cell differentiation, cell movement, cell size and shape, cell physiology, 
apoptosis, abnormal cell cycle, abnormal phenotypes, or general human 
physiology, occurring in rotating culture systems or in actual 
microgravity. The normal mammalian hydrodynamic milieu differs from a 
quiescent microgravity like environment. In utero, cells are constantly 
moved, pulled or forced to settle in different directions depending on the 
position of the organism, i.e.; standing, sitting or lying down. Movements 
from walking, running, and breathing agitate the cells; thereby adding 
hydrodynamic shear. An intrauterine environment is characterized by cells 
developing in amniotic fluid under constant hydrodynamic shear, resulting 
in high mass transfer. The present invention more closely emulates the 
intrauterine hydrodynamic and gas mass transfer environment where 
positional changes, breathing, movement, and hemodynamic pulsations 
continuously disrupt boundary layers. At the same time, the maternal 
physiology closely regulates the intrauterine environment; facilitating 
normal growth of organs. Major changes in maternal physiology would elicit 
great concern for abnormal fetal organ development. Yet, the standard of 
tissue culture practice for those of ordinary skill in the art, is to 
allow the cell culture pH and oxygen to fluctuate beyond in vivo limits. 
The invention, herein disclosed, integrates a novel hydrodynamic cell 
suspension environment, with regulation of media repletion and pulsatile 
mass transfer or diffusive, to achieve three-dimensional tissue constructs 
suitable for transplantation. An automated tissue culture system that 
facilitates three-dimensional growth, and is disposable, fulfills a need 
for technology to engineer autologous tissue constructs, as well as grow 
cells for basic research. 
PRIOR ART 
Publications 
M. L. Lewis, et. al., "Growth and Maintenance of Anchorage Dependent Cells 
in Zero Headspace Bioreactor Systems Designed for Microgravity", 
Proceedings, Spacebound '87, First Canadian Workshop on R&D Opportunities 
on Board the Space Station, May 6-8, 1987. 
"Space Bioreactor Science Workshop" Proceedings of an August, 1985 workshop 
by NASA, ED: D. R. Morrison, Government publication: NASA Conference 
Publication 2485, December, 1987. 
Aunins, J. G., M. S. Croughan, D. I. C. J. Wang, and J. M. Goldstein, 
Engineering Developments in Homogeneous Culture of Animal Cells: 
Oxygenation of Reactors and Scale-up, Biotechnology and Bioengineering 
Symp, No. 17, 699-723, 1986. 
Spaulding, G. F., "Perfusion in a NASA Cell Culture System", In-Vitro, 
30A(3), Pt.2:52-53, 1994. 
Towe, B. C., Flechsig, S., and G. Spaulding (1996) A Recirculating-Flow 
Fluorescent Oxygen Sensor, Biosensors & Bioelectronics, 11(8):799-803. 
Peterson, J. I., R. V. Fitzgerald, D. K. Buckhold (1984) Fiber-Optic Probe 
for in Vivo Measurement of Oxygen Partial Pressure, Anal. Chem., 56:62-67. 
Peterson, J. I., Goldstein, S. R., and R. V. Fitzgerald (1980) Fiber Optic 
pH Probe for Physiological Use, Anal. Chem. 52:864-869. 
Prior art includes the following patents and, the references cited therein, 
and are also incorporated herein fully as if set out verbatim. 
U.S. Pat. No. 5,527,705 issued to E. F. Mussi and H. E. Gray on Jun. 18, 
1996, is an example of a two chamber roller bottle. The chambers are 
coaxially aligned and separated by a microporous structure for the 
physical separation of two cell populations, one in each chamber. Both 
chambers have a gas headspace and are coaxially aligned. In contrast, the 
present invention has a zero headspace cell culture chamber in 
communication with a second chamber having a gas headspace, and a 
partition segments the roller bottle into two cylindrical chambers. The 
two chambers are contiguous through a hole in the center of the partition. 
U.S. Pat. No. 5,010,013 issued to J. M. Serkes et. al., on Apr. 23, 1991, 
is an example of a cell culture roller bottle with increased surface area 
for cell attachment. Corrugated channels were designed to add interior 
surface area for cellular attachment. The roller bottle teaches against 
cell suspension. It is of signal chamber design with a gas head space. 
U.S. Pat. No. 5,449,617 issued to F. W. Falkenberg, et. al., Sep. 12, 1995, 
relates to a roller bottle with a dialysis membrane separating two 
chambers. Both chambers have a gas headspace. The separating membrane is 
impermeable to liquids and microorganisms. In contrast, the present 
invention has a zero headspace cell culture chamber in communication with 
a second chamber having a gas headspace, and a partition segments the 
roller bottle into two cylindrical compartments--both chambers are 
hydrodynamically coupled. 
U.S. Pat. No. 3,847,749 issued to R. E. Smith, et. al., Nov. 12, 1974, 
relates to the automation of roller bottle feeding. Control elements are 
external to the roller bottle. In contrast, the present invention has 
internal control elements. 
U.S. Pat. No. 5,523,228 issued to M. Ingram, G. F. Spaulding, et. al., Jun. 
4, 1996, relates to a single chamber zero headspace gas permeable 
horizontally rotating cell culture bag. The bag is compliant. Oxygenation 
is through an air-membrane-fluid interface, and therefore gas mass 
transfer is substantially governed by the thickness and the diffusivity 
coefficient of the membrane oxygenator (J. G. Aunins, et.al., 1986); and 
boundary layer gradients. In contrast, the present invention has a zero 
headspace cell culture chamber in communication with a second chamber 
having a gas headspace, and a partition segments the roller bottle into 
two cylindrical compartments--both chambers are hydrodynamically coupled. 
U.S. Pat. Nos. 5,589,112 and 5,330,908 issued to G. F. Spaulding, and U.S. 
Pat. Nos. 5,026,650, 5,153,131, 5,437,998 and 5,665,594 relate to a gas 
permeable horizontally rotating microgravity cell culture vessels with 
zero headspace and solid body rotation. Solid-body horizontal rotation 
with zero headspace establishes a 1:1 viscous coupling of the culture 
media with the vessel wall, thereby eliminating boundary layers of 
gradient hydrodynamic shear (G. F. Spaulding, In-Vitro, 1994, 
30A(3),Pt.2:52-53). Oxygenation is through an air-to-membrane-to-fluid 
interface, and therefore gas mass transfer is substantially governed by 
the thickness and the diffusivity coefficient of the membrane oxygenator; 
and boundary layer gradients (J. G. Aunins, et.al., 1986). They teach 
against turbulence. In contrast, the present invention teaches turbulence, 
teaches hydrodynamic gradients, teaches tangential cell motion with media 
exchange, teaches oxygenation by direct contact of the gas with the media, 
and teaches a gas headspace. 
U.S. Pat. No. 5,057,428 issued to S. Mizutani et. al. on Oct. 15, 1991, 
relates to a cylindrical bioreactor tank which is rotated about a 
horizontal axis. There is a cylindrically shaped mesh in the chamber, 
which defines inner and outer chambers. Pipes conveys oxygen from an air 
pump into the chamber and a flow path is established to flow return pipes 
which provide for continuous replenishment of spent media. It is 
complicated to assemble and disassemble. 
U.S. Pat. No. 5,015,585 issued to J. R. Robinson, on May 14, 1991, 
discloses a bioreactor construction utilizing a single polymer in a 
concentric geometric configuration to add durability and reduce 
complexity. 
U.S. Pat. No. 3,821,087 issued to R. A. Rnazek et. al., on Jun. 28, 1974, 
discloses a cell growth system where cells are grown on membranes in a 
nutrient medium. Nutrient fluids carrying oxygen flow through the roller 
bottle and pass through a membrane to contact the cell culture. The fluid 
is driven by an impeller into the culture roller bottle. Numerous 
capillaries are used to distribute oxygen and nutrients over a large area 
to reduce uneven distribution of resources. There is no rotation of the 
roller bottle. It is complicated to assemble and disassemble. 
U.S. Pat. No. 4,749,654 issued to D. Karrer et. al., on Jun. 7, 1988, 
relates to a cell growth system using gas permeable membranes and a waste 
gas removal system. A stirrer is used for agitation. Oxygen flows in 
through one side of the membrane and carbon dioxide flows out the other. 
U.S. Pat. No. 4,948,728 issued to G. Stephanopauous et. al., on Aug. 14, 
1990, discloses a porous ceramic material with a plurality of flow 
passages. A biofilm is in contact with an inner wall and a gas permeable 
membrane covers the outer wall. An oxygen flow along the outer wall 
permeates the membrane and ceramic housing to reach the biomaterial. 
Nutrients flow along the inner wall in direct contact with the biofilm. 
There is no rotation of the roller bottle. 
U.S. Pat. No. 4,962,033 issued to J. M. Serkes et. al., on Oct. 9, 1990, is 
an example of a cell culture roller bottle with increased surface area for 
cell attachment. Corrugated channels were designed to add interior surface 
area for cellular attachment. In contrast to the present invention in 
which there is a zero headspace cell culture chamber in communication with 
a media containing chamber that has a gas headspace; the roller bottle has 
a single compartment with a gas headspace. The roller bottle teaches 
against cell suspension. 
U.S. Pat. No. 4,391,912 issued to K. Yoshida et. al., on Jul. 5, 1983, is 
an example of a cell culture system with hollow fibers that is not 
rotated. 
U.S. Pat. No. 5,496,722 and 5,637,477 issued to G. F. Spaulding et. al., 
relate to culturing in NASA microgravity culture vessels in solid-body 
rotation with zero headspace. Solid-body horizontal rotation with zero 
headspace establishes a 1:1 viscous coupling of the culture media with the 
vessel wall, thereby eliminating boundary layers of gradient hydrodynamic 
shear (G. F. Spaulding, In-Vitro, 1994, 30A(3),Pt.2:52-53). Oxygenation is 
through an air-to-membrane-to-fluid interface, and therefore gas mass 
transfer is substantially governed by the thickness and the diffusivity 
coefficient of the membrane oxygenator; and boundary layer gradients (J. 
G. Aunins, et.al., 1986). We did not maintain a physiological pH. We 
taught against turbulence. In contrast, the present invention teaches 
turbulence, teaches hydrodynamic gradients, teaches tangential cell motion 
with media exchange, teaches oxygenation by direct contact of the gas with 
the media, and teaches a gas headspace. The novel hydrodynamics and novel 
culture environment yields cellular constructs with unique phenotypes. 
U.S. Pat. No. 5,567,598 issued to D. T. Stitt et. al., on Oct. 22, 1996, 
discloses the use of ruthenium diimine complexes for the detection and 
evaluation of metabolic activity of microorganisms based on their ability 
to consume dissolved oxygen. A ruthenium diimine complex was used in a 
t-flask to measure E. coli growth. They did not teach monitoring of tissue 
metabolism, the sensor and instrument adaptations required for monitoring 
a sensor in rotation were not taught, and they did not teach oxygen 
regulation based on sensor data. The current invention improves the prior 
art by adapting a ruthenium diimine complex based sensor and monitoring 
instruments to a rotating culture vessel, and utilizes the adaptation for 
monitoring and controlling dissolved oxygen. 
Towe, B. C., Flechsig, S., and G. Spaulding (1996) A Recirculating-Flow 
Fluorescent Oxygen Sensor, Biosensors & Bioelectronics, 11(8):799-803. We 
disclosed the characteristics of a liquid ruthenium diimine mixture for 
dissolved oxygen sensing. We taught against solid phase sensors. 
Peterson, J. I., R. V. Fitzgerald, D. K. Buckhold (1984) Fiber-Optic Probe 
for in Vivo Measurement of Oxygen Partial Pressure, Anal. Chem., 56:62-67. 
They teach optical sensing of oxygen. They did not teach monitoring of 
tissue culture metabolism, the sensor and instrument adaptations required 
for monitoring a sensor in rotation were not taught, and they did not 
teach oxygen regulation based on sensor data 
Peterson, J. I., Goldstein, S. R., and R. V. Fitzgerald (1980) Fiber Optic 
pH Probe for Physiological Use, Anal. Chem. 52:864-869. They teach optical 
sensing of pH and the use of phenol red. They did not teach monitoring of 
tissue culture metabolism, the sensor and instrument adaptations required 
for monitoring a sensor in rotation were not taught, and they did not 
teach pH regulation based on sensor data 
SUMMARY OF THE PRESENT INVENTION 
In the present invention, the culture vessel is embodied in a roller bottle 
having an open first end and an enclosed second end. The open first end is 
used to introducing and remove media and is large. Said large opening 
enables the removal of large tissue constructs--in contrast to t-flasks 
and syringe ports. A means for retaining the culture media is disposed to 
said first end. Said mean for media retention is disposed to said first 
end to form a substantially fluid tight seal. Said means for retaining 
culture media can be a commercially obtained cap, as is know in the art. 
The roller bottle is partitioned into two chambers, a first chamber and a 
second chamber. Said first chamber is the media chamber; and is defined by 
said first end with means for media retention, and a partition. Said 
second chamber is the tissue culture chamber; and is defined by said 
partition and said enclosed second end. Both first and second chambers are 
contiguous through an opening in said partition. Said opening in said 
partition is centered with the central annular axis of the roller bottle. 
Said opening in said partition is smaller than the inner diameter of the 
roller bottle, i.e.; the area of the opening is less than the area the 
partition subtends in the roller bottle. 
In constructing the roller bottle, the roller bottle is constructed of 
materials that are suitable for cell and tissue culture. Said materials 
are known in the art and can be chosen from; polystyrene, glass, 
polyethylene, polysulfone, methyl methacrylate, high density polyethylene, 
low density polyethylene, polyethylene terephthalate glycol, 
perfluoroalkoxy, polycarbonate, polyvinylidene fluoride, 
polytetrafluoroethylene, ultra high molecular weight polyethylene, nylon, 
Teflon.RTM., crystalline polystyrene, metallocene-based polypropylenes, 
syndiotatic polystyrene, and the like. In one embodiment, said roller 
bottle is constructed as a single unit, by injection molding or blow 
molding or extrusion, out of crystalline polystyrene, said walls defining 
said roller bottle being substantially impermeable to gas and liquid 
exclusive of said opening in said partition. In an alternate embodiment, 
said roller bottle is the assemblage of two members. A first cylinder 
member having an open first end and an enclosed second end; and, a second 
cylinder member having an open first end and an enclosed second end. Said 
second end of said second cylinder member defines said partition. Said 
second cylinder member is inserted into said first cylinder member whereby 
said partition and said enclosed second end of said first cylinder member 
defines the second chamber (tissue culture chamber). Said second cylinder 
member and cap means for retaining fluid defines said first chamber (media 
chamber). Said cap means is disposed to said first cylinder member in a 
fashion to secure said second cylinder member within said first member and 
retain media. 
In constructing said means for retaining media, the fluid retention means 
is constructed of materials that are suitable for cell and tissue culture. 
Said materials are known in the art, and can be chosen from: polystyrene, 
glass, polyethylene, polysulfone, methyl methacrylate, high density 
polyethylene, low density polyethylene, polyethylene terephthalate glycol, 
perfluoroalkoxy, polycarbonate, polyvinylidene fluoride, 
polytetrafluoroethylene, ultra high molecular weight polyethylene, nylon, 
Teflon.RTM., crystalline polystyrene, metallocene-based polypropylenes, 
syndiotatic polystyrene, and the like. In one embodiment, said fluid 
retention means is a cap constructed of optically transparent crystalline 
polystyrene, and secured to the roller bottle by screw threads therein 
forming a substantially fluid tight seal. Said roller bottle having a 
closed environment substantially free from gas exchange. 
In a alternate embodiment for an open environment for gas exchange, said 
cap is comprised of microporous polymeric materials with high liquid 
intrusion pressures (e.g., Pall Specialty Materials, N.Y.). Said 
microporous materials enables enhanced gas exchange between the gas 
headspace in the first chamber and the ambient external air. Said gas 
exchange would be of higher flux, with respect to gas-to-membrane-to-fluid 
exchange, because it is known in the art that gas-to-membrane-to-gas 
exchange has a higher rate of transfer because of the lower solubility of 
gas in liquid. Therefore, gas exchange through a liquid-membrane-liquid 
process is slower than a gas-membrane-gas process. Herein disclosed as a 
novel means for oxygen transfer to a gas headspace chamber. Oxygenation 
from ambient air-to-membrane-to-gas headspace then from gas 
headspace-to-media from said first chamber to said second chamber provides 
for greater gas mass transfer--a novel gas mass transfer process. Suitable 
microporous materials are known in the art and are commercially available 
in a variety of sizes and materials. Materials for microporous cap 
construction can be chosen from: polystyrene, glass, polyethylene, 
polysulfone, methyl methacrylate, high density polyethylene, low density 
polyethylene, polyethylene terephthalate glycol, perfluoroalkoxy, 
polycarbonate, polyvinylidene fluoride, polytetrafluoroethylene, ultra 
high molecular weight polyethylene, nylon, Teflon.RTM., crystalline 
polystyrene, metallocene-based polypropylenes, syndiotatic polystyrene, 
and the like. 
In the alternate embodiment, microporous materials can include microporous 
membranes that are known in the art and are commercially available as 
screw on caps having a microporous membrane disposed to the cap that 
allows gas exchange yet retains fluid. In the art there are two problems 
with gas exchange membranes that are in direct contact with a zero 
headspace culture chambers. 1) A cell coming in direct contact with a gas 
exchange membrane may become toxic because of the high oxygen 
concentration at the interface; 2) The materials used to construct the 
membrane are often toxic; and 3) When there is a gas headspace on one side 
of a gas exchange membrane, and media having zero head space on the other 
side, media is lost by evaporation. Media evaporating from the zero 
headspace side to the gas headspace side, lowers the pressure on the media 
side. Eventually, when the pressure is low enough, gas is drawn from the 
gas headspace side through the membrane to the media side, where the gas 
forms micro-bubbles at the membrane-fluid interface. With continued vapor 
loss, micro-bubbles can become larger and dislodge from the membrane, 
coalescing at the top of the culture chamber, therein causing increased 
shear in a horizontally rotating culture vessel. The invention herein 
disclosed, overcomes the prior art difficulties by establishing a novel 
gradient that favors bubble dissolution in the cell culture chamber (zero 
headspace, said second chamber). 
In culturing tissue, the culture roller bottle is sterilized and fresh 
culture media, cells and/or tissue are admitted to completely fill the 
tissue culture chamber (said second chamber) without any bubbles, i.e.; 
zero headspace. Culture media without cells or tissue is admitted to the 
media chamber to fill approximately 2/3 of the media chamber volume (said 
first chamber). The remainder of the media chamber is filled with gas. A 
cap or other means to retain media is disposed to the roller bottle. The 
roller bottle is placed in a horizontal position. In placing the roller 
bottle in a horizontal position, the gas bubble in the media chamber 
becomes trapped at the top half of the media chamber (said first chamber) 
and above said opening in said partition. The gas volume is chosen so that 
the bubble is not so large as to move through said opening in said 
partition and enter said tissue culture chamber (said second chamber). A 
zero headspace second chamber and gas headspace first chamber establishes 
a hydrostatic gradient from said second chamber to said first chamber, 
whereby said media in said second chamber is held at a higher fluid level 
than said first chamber (when placed in a horizontal position). Adhesive 
and cohesive liquid forces hold said media at a higher level in said zero 
headspace second chamber. Consequently, a hydrostatic gradient is formed 
from said second chamber, where the fluid level in a horizontal position 
is highest, and said first chamber where the gas bubble displaces said 
media at the top of that chamber. Herein disclosed as a novel cell culture 
environment embodied in a culture vessel characterized by a novel 
hydrostatic gradient from said zero headspace chamber to said gas 
headspace chamber. 
In establishing a gas gradient, said first chamber has a gas headspace and 
said second chamber has zero headspace. It is commonly known in the art 
that the gas solubility coefficient is higher for gas than it is for 
liquid. Therefore, the dissolved gas gradient, augmented by cellular 
metabolism, favors the dissolution of bubbles in said zero headspace 
chamber and the re-equilibration with the gas headspace (or the 
transposition of the gas to non-gaseous metabolites by cellular 
metabolism). Continuous dissolution of undissolved gas in said second 
chamber, helps to remove gas bubbles in that chamber and maintain a zero 
headspace. Herein, disclosed as a novel means to remove gas bubbles from a 
zero headspace cell culture chamber by establishing a dissolution gradient 
from said zero headspace chamber through said partition to said first 
chamber having a gas headspace. 
In rotating the vessel, culture media in said media chamber covers the 
opening in said partition both when the roller bottle is standing 
vertically, and when it is lying horizontally. In a horizontal position, 
the roller bottle containing media and cell and/or tissue are disposed to 
a means for horizontal rotation. Said means are known in the art. Cells 
and tissue constructs are suspended in said tissue culture chamber one by 
a combination of turbulent mixing and sedimentation, when in horizontal 
rotation. As the roller bottle is horizontally rotated, turbulence from 
said first chamber through said opening in said partition is coupled to 
said second chamber, and is blended with the turbulence caused by the less 
than 1:1 viscous coupling of the roller bottle wall with the culture media 
in said first and second chambers. The novel blend of turbulence 
facilitates three-dimensional cell and tissue growth, and enhances mass 
transfer. The tissue constructs are unique due to their derivation in a 
novel hydrodynamic culture environment. Cell culture fidelity is enhanced 
by adding turbulence, enhancing mass transfer esp., oxygen, and better 
regulating the environmental parameters. In the present invention, the 
novel culture environment yields tissue constructs that are of higher 
quality then described in the prior art. The tissue constructs are formed 
under a novel combination of hydrodynamic selection pressures; turbulence, 
shear, augmented mass transfer, suspension, narrower environmental 
extremes and pulsatile mass transfer when cells pass by said opening 
through said partition. 
In preventing tissue constructs from moving between said first and second 
chambers, velocity vectors are imposed on said constructs that alleviate 
movements through said opening in said partition. When the culture vessel 
is in the vertical position, cells sediment to the bottom i.e., said 
enclosed end of said second chamber, and away from said opening in said 
partition. In horizontal rotation, the major velocity vectors for said 
suspended constructs are towards earth. Hence, the movement of suspended 
constructs is tangential to said opening in said partition. Tangential 
movement without velocity vectors in the direction of the annular axis 
creates a barrier to entry without the use of potentially destructive 
mechanical or physical barriers. Herein, disclosed as a novel means to 
constrain the movement of cells and tissue constructs. 
In rotating the tissue constructs, the roller bottle and means to retain 
media (e.g.; cap) are disposed to a horizontally rotating drive means. 
Drive means are known in the art and are available commercially, listed in 
many catalogs. Rotating speeds can range from 0 to 50 RPM and are 
determined by an operator with ordinary skill in the art, and are based on 
the degree of aggregation and turbulence required for the particular 
experiment. 
In generating hydrodynamic turbulence and shear, both first and second 
chambers are contiguous through an opening in the partition. On one side 
of said partition, in said first chamber, there is a combination of gas 
and culture media. On the other side of said partition, in said second 
chamber, there is media containing cells or tissue with zero headspace. 
Novel hydrodynamics are created in the culture roller bottle by breaking 
the solid body rotation of said second culture chamber through the 
coupling with the turbulence from gas/media rotation in said first 
chamber. Hence, said second chamber loses the 1:1 viscous coupling, 
therein generating wall turbulence. Turbulence is generated by viscous 
slip at the wall-media interface. Turbulent eddies resulting from the 
viscous slip in said second chamber, impart shear to the growing tissue. 
It is known in the art that shear leads to increased mass transfer. Both 
shear and increased mass transfer facilitates cellular differentiation. 
The present inventions discloses novel hydrodynamics that uniquely blends 
both shear and increased in mass transfer. The combination improves the 
fidelity of the growing tissue construct and enables unique phenotypes. 
In oxygenating the media and tissue construct, cell growth is maintained at 
a distance from the gas-media interface. At the gas-media interface, 
oxygen levels are often higher than those observed in vivo. Oxygen and 
other nutrients are exchanged at said opening in said partition, away from 
the gas-liquid interface, therein limiting oxygen toxicity. As the tissue 
constructs sediment, they pass through areas of high and lower oxygen 
tension, and nutrient concentration. Discontinuous oxygenation combined 
with the novel hydrodynamics of this invention yields novel tissue 
constructs especially suited to transplantation. Cell sedimentation 
through areas in the media containing high dissolved oxygen and low 
dissolved oxygen result in pulsatile exposure, a modeling of normal in 
vivo hemodynamic mass transfer. In augmenting mass transfer, turbulence 
and shear are constituents of the novel hydrodynamic tissue culture 
environment. Both turbulence and shear augment mass transfer. In mammalian 
cells, oxygen is the rate limiting step. Enhanced gas mass transfer from 
turbulence and shear, support the engineering of higher density tissue 
constructs. Tissue constructs are exposed to high mass transfer at two 
locations in the tissue culture chamber; at the media-wall interface where 
fluid streamlines are turbulent, and during suspension when tangentially 
passing said opening in said partition. Hence, tissue constructs growing 
in the novel hydrodynamic environment of this invention, pass through 
regions of high mass transfer followed by regions of lower mass transfer. 
Periods of high mass transfer followed by periods of lower mass transfer 
is a rudimentary simulation of in vivo hemodynamic systole followed by 
diastole. In vivo, during systole there is high mass transfer followed by 
diastole where mass transfer is lower. 
In preconditioning islets to lower oxygen tension, the oxygen in the 
culture environment is regulated at a level found in the transplantation 
site. In contrast to naturally growing tissue having an internal blood 
supply, tissue for transplantation must obtain nutrients by diffusion from 
the outside of the construct to the interior. Most in vitro derived tissue 
transplants fail due to an inadequate blood supply. The present invention 
is useful for preconditions tissue constructs to lower dissolved oxygen 
levels. This invention teaches control and regulation of the dissolved 
oxygen culture environment in parallel with the engineering of 
3-dimensional tissue constructs. Tissue constructs can be proliferated and 
differentiated in a low oxygen tension environment. Reduced oxygen tension 
goals are based on the calculated oxygen tension that the tissue construct 
is expected to be exposed to in the transplantation site. 
In an embodiment for pancreatic islets, encapsulated for transplantation: 
It is not generally known that isolated islets are hypoxic, using any 
isolation technique. Standard insulin staining of the isolate often 
demonstrates a cherry red population that is inferred to be a fully 
functional population (together with viability stains). However, the 
hypoxic insult of isolation triggers apoptosis resulting in programmed 
cell death, and core necrosis. In vivo, the islet is highly vascularized 
often having a capillary vascular supply devoted to each islet. Isolation 
of that islet severs the vascular supply leading to hypoxia. It has been 
incorrectly assumed that by suspending the islet isolate in oxygenated 
cell culture media, oxygenation would be fully restored. The peripheral 
cells (on islets larger than 150.mu. diameter) impose a diffusive gas mass 
transfer barrier that lowers the mass transfer rate to below 
5.times.10.sup.-12 gms O.sub.2 per cell per hour. Encapsulating the islets 
imposes a second oxygen diffusivity barrier. The encapsulating material 
reduces perfusion over the cell membrane surfaces, thereby reducing the 
diffusivity constant to near passive diffusivity levels--a function of 
thickness, and pore size and porosity. Consequently, boundary conditions 
are established at the capsule surface (as opposed to the cell membrane 
surface), resulting in a zone of oxygen depletion that surrounds the 
islet. Additionally, cells in the hypoxia induced apoptotic or necrotic 
core eventually die, resulting in the release of autolytic enzymes. 
Autolytic enzymes concentrate in the capsule, further degrading viability. 
Foreign antigens are released by cell death. They contribute to the 
foreign antigen pool that provokes a immunological response by the host to 
the encapsulated transplant. The net result is generally a 90% reduction 
in function within a few weeks after the transplant. The instant invention 
preconditions developing islet constructs to low oxygen therein reducing 
cellular death, increasing transplantation viability, and reduces the 
release of cellular antigens that can induce cytokine release which can 
provoke an inflammatory response. 
In aseptically monitoring the cell and tissue culture environment, sensors 
are positioned in said second chamber in contact with said culture media. 
Said sensors have properties, whereby their fluorescent intensity and/or 
color changes in response to an analyte in said culture media. Said 
properties include a dye embedded in a polymeric matrix or attached to a 
surface and having a fluorometric and/or calorimetric change in response 
to an analyte. Fluorescent changes and/or colorimetric changes and the 
analyte that they respond to, are known in the art. Said 
fluorescent/colorimetric change, in response to said change in an analyte 
in said media, can be monitored optically. There are advantages to 
embedding said dye in said sensor and disposing said sensor on said roller 
bottle wall in contact with said media. The first advantage is, by 
depositing a controlled amount of sensor material on to said wall of said 
roller bottle, the concentration of said dye in said sensor is known. 
Knowing said dye concentration is important in determining the 
concentration of an analyte in said media i.e., the 
fluorometric/colorimetric change is proportional to the dye and analyte 
concentration. A second advantage is that said fluorometric/colorimetric 
changes can be monitored externally through said wall of said roller 
bottle. Such monitoring device incorporates an LED, or laser diode, to 
interrogate the sensor through the vessel wall, two photodiodes to monitor 
LED output and sensor absorption, a 16 bit analog-to-digital converter for 
each photodiode, and a microcontroller to synchronously modulate and 
demodulate the signal to remove background noise and synchronize sensing 
with rotation. The microcontroller regulates the electromagnet control of 
the control rod that occludes or allows mixing through said opening in 
said partition, therein providing environmental regulation e.g., pH and 
oxygen. Hence, changes in said sensor can be monitored and controlled 
externally, therefore the roller bottle sterility need not be breached. In 
the preferred embodiment, said sensor is disposed to said wall of said 
roller bottle during manufacturing, and said sensor is sensitive to pH or 
dissolved oxygen. The instant invention herein disclosed embodies a 
significant improvement in cost, reliability and sterility, as will be 
recognized by those skilled in the art. 
In monitoring and regulating the culture environment, information on a 
particular analyte is gather from said external monitors, interrogating 
said internal sensors for fluorometric and/or colorimetric changes 
resulting from changes in said analyte concentration. Information from 
said interrogation is utilized to control the mixing between said first 
and second chambers. Said control of said mixing is accomplished by a 
control rod that occludes or does not occlude said opening in said 
partition. Said control rod is constructed of a polymeric material and is 
disposed in said first chamber. Said control rod further includes a 
north-south magnet encased in said polymeric material previously listed 
and having said north-south poles coaxially orientated with said annular 
axis of said roller bottle. Suitable magnets are commercially available 
and know in the art. An electromagnet is positioned external to said 
roller bottle, juxtaposition to said means to retain fluid, and having 
poles coaxially aligned with said annular axis. In controlling occlusion, 
said electromagnet is energized, having said pole juxtaposition to said 
means for retaining fluid. Energizing said electromagnet with one polarity 
generates a magnetic field that is the same polarity as the nearest pole 
of said magnet in said control rod. Generating a similar magnetic field to 
said nearest pole of said control rod, magnetically repels said control 
rod away from said means to retain fluid and into a position that occludes 
said opening in said partition. Conversely, reversing said electromagnetic 
field draws said pole of said control rod away from said opening in said 
partition, wherein mixing occurs between said first and second chambers. 
It will be appreciated by those skilled in the art that the invention 
herein disclosed is low cost and does not breach sterility. Moreover, the 
instant invention enables better control of the cell and tissue 
environment for better engineering of tissue constructs. Said improved 
control is used to tailor the engineering of novel tissue constructs. 
In achieving validation, proliferating constructs must be monitored and 
documented. Food and Drug Administration (FDA) validation and lot release 
of tissue constructs, mandates numerous test of the tissue constructs for 
sterility, reproducibility, and fidelity. Monitoring and controlling said 
cell and tissue culture environment alleviates batch testing and replaces 
post hoc analysis with real time data. For example; once a growth profile 
for a tissue construct is established, the rate and time of change for the 
monitored parameters (e.g., pH, oxygen, CO.sub.2, lactic acid, glutamine, 
ammonia, urea, nitrogen, glycosylation linkages--all potential analytes to 
be monitored by said sensors) define the growth and development of the 
required tissue construct. Deviations from the normal profile indicate 
aberrancy. Aberrance can translate into loss of sterility, 
reproducibility, and/or fidelity. Matching the normal profile can be used 
as validation method to fulfill the FDA lot release requirements. It will 
be appreciated by those skilled in the art, that real time validation 
substantially reduces tissue engineering costs, and environmental control 
enhances the reproducibility. 
The present invention discloses a novel fluid dynamic environment embodied 
in a tissue culture roller bottle with two chambers, processes for 
engineering unique 2-dimensional and 3-dimensional tissue constructs, and 
uniquely segregated cell constituents of a three-dimensional tissue 
construct i.e., segregated by novel hydrodynamic selection pressures and 
environmental control. Enhanced gas mass transfer supports the maintenance 
of higher density tissue constructs. Monitoring and control automates the 
culture process and improves fidelity while lowering costs. It will be 
appreciated that the tissue culture roller bottle is simple to construct, 
very low cost, can be widely used by existing laboratories with 
conventional equipment, and is suited to autologous tissue culture.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
Referring now to FIG. 1, where the perspective view shows the general 
organization of the present invention. The tissue culture roller bottle is 
divided by a partition 5 into two chambers; a first chamber 6 for culture 
media, and a second chamber 7 for a mixture of culture media and cells 
and/or tissue. The media chamber 6 and the tissue culture chamber 7 are 
contiguous through an opening 4 in the partition 5. The roller bottle is 
constructed of a polymer suitable for tissue culture. 
Referring now to FIG. 2, where the general organization of the present 
invention is schematized. A means to retain culture media, e.g., 
conventional cap 1, is disposed to said first end of said tissue culture 
roller bottle. Culture media 3 is present in said media chamber 6 and in 
said tissue culture chamber 7. The gas bubble 2 in said media chamber 6 
floats to the highest point in media chamber 6 and above said opening 4 in 
said partition 5. Media 3 between said two chambers is contiguous through 
said opening 4 in said partition 5, and covers up said opening 4 in said 
partition 5. Therefore, said gas bubble 2 in said media chamber 6 is 
prevented from entering said tissue culture chamber 7. 
Referring now to FIG. 3, where the general organization of the automated 
version of the present invention is schematized. A means to retain culture 
media, e.g., conventional cap 1, is disposed to said first end of said 
tissue culture roller bottle. In said media chamber 6 there is a control 
rod 11 constructed of a polymer suitable for tissue culture. Encased in 
said control rod 11 is a north south magnet 10. Said controller rod 11 
with its encased magnet 10 is mechanically constrained to the central 
annular axis of said roller bottle. Said constraints can be but are not 
limited to, posts or other mechanical means 8 deposed to said partition 5 
or said control rod 11. Alternatively, said control rod 11 with said 
magnet encased 10, can be held by independent mechanical means. Said 
mechanical means 8 for constraining said control rod 11 are constructed of 
polymers suitable for tissue culture. When said electromagnet 9 is 
energized, said control rod 11 is repelled or attracted to said 
electromagnet because of said encased north south magnet 10. Said 
electromagnet 9 is located outside of said roller bottle. One polarity 
will cause said electromagnet 6 to repel said control rod 11, the opposite 
polarity will result in attracting said control rod 11. Repelling said 
control rod 11 occludes said opening 4 in said partition 5. 
Electromagnetic attraction of said control rod 11 opens said port 4 in 
said partition 5. External electromagnetic control 9 of said internal 
control rod 11 achieves several advantages: Sterility is not breached 
during the controlling actions, movement is externally regulated, said 
motion of said control rod 11 imparts turbulence and directs the 
turbulence, mixing between said two chambers is regulated, and the 
regulation of the mixing between said two chambers 6 and 7 can be based on 
said sensor data 12 obtained in said culture chamber 7. Said sensor 12 is 
disposed to said wall of said culture chamber 7. Maintained in said sensor 
12 are dyes or other suitable indicators know in the art for detecting 
constituents in said culture media. Indicator dyes can be calorimetric or 
fluorescent, for example phenol red and ruthenium diimine complexes. In 
particular, the ruthenium diimine complexes can be chosen from 
tris-2,2'-bipyridyl ruthenium (II) salts, including the chloride 
hexahydrate salt, tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) 
salts, including the chloride salt, and 9,10-diphenyl anthracene. To 
detect the changes in said sensor 12, a detector suitable for said 
particular sensor is located externally. It will be appreciated by those 
of ordinary skill in the art that said culture roller bottle with sensor 
12 and control rod 11 are simple to construct, low cost, and disposable. 
Moreover, a variety of features are integrated into a single unique 
disposable roller bottle. 
As an example of use of said cell culture system, Ham's F10 media can be 
sterilized and placed into said cell culture chamber. Said tissue culture 
chamber is completely filled with cells and/or tissue and media. Cells are 
commercially available, derived from cell lines, derived from animals, or 
obtained from human donors. Herein, cells shall refer to cells, tissue or 
a mixture of cells and tissue. Said media chamber is then filled to 2/3 
its volume with culture media, the remaining 1/3 volume is gas. Chamber 
volumes can range from less that 1 ml to several litters. To provide the 
necessary ambient environment, said culture roller bottle is placed into a 
conventional incubator where it rotates, and turbulence, pulsatile mass 
transfer and shear provide for proliferation and monolayer and/or 
three-dimensional growth. 
Cells are suspended by adjusting the rotating speed. Suspension is observed 
when there is a balance between sedimenting and turbulent forces. In the 
present invention, mammalian and animal cells will grow and aggregate in 
suspension. Cellular aggregates can range from 50 microns to several cm. 
As the cells aggregate, they form an autologous extracellular matrix upon 
which cells adhere, differentiate and become 3-dimensional tissue. Tissue 
constructs developed in the novel hydrodynamic environment of this 
invention are unique. Turbulence, pulsatile mass transfer, and shear place 
unique selection pressures upon the growing construct. Cells that are 
robust to turbulence and shear, thrive on the outside of the construct 
where the exposure is the greatest. Those cell subpopulations that require 
lower oxygen tension thrive in the core of the construct. Consequently, 
selection and segregation based on the novel culture and hydrodynamic 
environment leads to the growth and differentiation of tissue constructs 
that are unique to the two-chamber culture environment of this invention. 
The present invention improves the gas mass transfer to the culture media, 
and eliminates the toxic silicone membrane oxygenator that is in general 
use in the art. Gas in said media chamber is in direct contact with 
culture media. As said roller bottle rotates, the gas in said media 
chamber is directly mixed with culture media. Rotation disrupts the 
gas-media boundary layers, facilitating gas mass transfer. Media adherent 
to said wall and drips off the walls during rotation, further augmenting 
gas mass transfer. Elimination of the silicone membrane (or any other 
oxygenation material) oxygenator removes the major impediment to gas mass 
transfer. Moreover, silicone has been implicated as a potential disease 
causing agent. It is known in the art that during periods of cellular 
proliferation and differentiation, cell are especially susceptible 
damaging agents. Engineering tissue constructs requires cellular 
proliferation and differentiation. 
The present invention mitigates the loss of cells during feeding. Examples 
of culture vessels that lose cells during media replacement are: t-flasks, 
petri dishes, roller bottles, stirred vessels, and horizontally rotating 
vessels. In the present invention, while in vertical position, cell 
sediment to the bottom of the tissue culture chamber. Media is siphoned 
from the surface without disturbing the sedimented cells. In contrast to 
t-flasks and rotating vessels, where the vessel is place on end and 
non-adherent cells are lost when the media is removed. In horizontal 
rotation of the present invention, cell movement is tangential to the 
opening in the partition. Consequently, cells fall past the opening. The 
velocity vectors direct cellular movement past the opening thereby 
restricting their entrance into the media chamber. To replace media in the 
media chamber, the vessel it tilted to a vertical position wherein the 
cells sediment to the bottom of the tissue culture chamber and away from 
the media chamber. Thus, substantially all the cells are retained during 
media repletion/replacement. Following media repletion, the vessel is 
gently tipped back to a horizontal position and rotated. During rotation 
the cell movement vectors again become tangential to the opening in the 
partition. It will be appreciated by those of ordinary skill in the art, 
that media exchange at the opening in the partition without cellular loss 
to the media chamber, is an instant invention. Furthermore, the tissue 
constructs are unique because they are engineered from cell subpopulations 
that are typically lost during conventional media repletion. The lost 
subpopulation includes stem cells. The aggregation of cells that are 
typically lost during media repletion with cells that are generally 
present after media repletion, yields unique tissue constructs. The unique 
constructs have phenotypes that differ from constructs cultured in other 
vessels. 
EXAMPLE 1 
A tissue culture roller bottle of this invention, injection molded with 
cell culture grade polystyrene (e.g., Wheaton Science Products, MilIville, 
N.J.), was sterilized by methods know in the art. The roller bottle was 
placed in a vertical position. A mixture of culture media and cells was 
added until the culture chamber was completely filled. Cell concentrations 
ranged from 10.sup.5 cells/ml to 10.sup.7, and were normal human epidermal 
keratinocytes. Other cells in the co-culture included; fibroblasts, 
melanocytes, and epidermal stem cells. Fresh culture media was utilized to 
fill 2/3 of the remaining volume of the media chamber. The last 1/3 volume 
of the media chamber was left empty i.e., gas filled the remaining 1/3 
media chamber volume. The cap was disposed to the first opening and the 
culture roller bottle was slowly tilted to a horizontal position. In 
moving the culture roller bottle from a vertical position, the gas trapped 
in the media chamber moved to the highest point in the media chamber. The 
media volume was chosen so that when the roller bottle was positioned 
horizontally, the culture media covered the opening in the partition, 
preventing the gas bubble from entering the tissue culture chamber. The 
culture roller bottle was disposed to a means to rotate, cell culture 
rotators are know in the art and can be purchased (e.g. Fisher Scientific, 
Tex.). The culture roller bottle disposed to a rotator was placed in a 
tissue culture incubator at 37.degree. C. Rotation was started at 
approximately 20 rpm. Small cell aggregates formed within a few hours and 
larger aggregates gradually develop thereafter. Microcarrier beads may be 
included for attachment and support, or the tissue culture chamber walls 
may be specially modified to encourage tissue attachment. Microcarrier 
beads with densities less than the cell culture media, can be utilized for 
attachment, and to constrain tissue constructs to the area surround the 
annular axis and away from the cylinder wall. 
Constructs remain undisturbed in culture for longer periods of time, as 
compared to conventional culture. The extended culture duration is due to 
the additional media contained in the media chamber. Mixing between the 
media chamber and the tissue culture chamber is slow. Slowly mixing fresh 
media from the media chamber, with the tissue culture chamber, effectively 
prolongs the undisturbed culture time. Standard culture practice is to 
re-feed cultures by replacing 50-100% of the depleted media with fresh 
media. A consequence of that approach, is that de novo growth factors are 
abruptly removed, harming the proliferating constructs. In the present 
invention, the rate of media repletion (growth factor reduction) is 
substantially reduced; and growth factor concentration remained higher 
throughout the culture process. 
The process of choosing a cell type(s) and proliferating the cells into 
3-dimensional aggregates, constitutes tissue engineering. Cellular 
aggregates derived from the present invention have broad utility, and 
special utility in autologous transplantation because of the ability to 
pre-conditioning explants. The novel combination of hydrodynamic forces 
and augmented mass transfer is suited to the growth of autologous tissue 
constructs and unique tissue constructs. Sizes and shapes vary and can 
take the form of cellular aggregates or, sheets of cellular and 
extracellular material. The present invention can be used to expand a 
population of normal human cells or stem cells into a larger population. 
That expanded cell population would be useful for transplantation back to 
the original host or to a different tissue compatible host. Additionally, 
the engineered tissue can be used for research; testing gene therapies, 
viral propagation, new drug treatments and/or for testing fundamental 
biological theory. 
EXAMPLE 2 
In an alternate embodiment for the manufacturing of the culture roller 
bottle, the media chamber and partition with opening were injected molded 
or vacuformed as a separate chamber member. The media chamber was inserted 
into the culture roller bottle member. The first media chamber member 
defined the media chamber, the volume between the partition and the second 
of the cylinder member defined the tissue culture chamber. 
EXAMPLE 3 
In an alternate embodiment for automating media repletion, the control rod 
member incorporated six leg members that extended from and perpendicular 
to the annular axis. The leg members mechanically constrained the control 
rod in the media chamber but allowed free mobility along the annular axis 
within the media chamber. The control rod member was centered in the 
annular axis of the cylinder. Encased in the control rod, was a 
north/south magnet, also aligned with the annular axis of the cylinder. 
External to the culture roller bottle was a conventional electromagnet or 
other means for generating a magnetic field. The preferred location was 
near the cap. When the electromagnet was energized and generated a 
magnetic field opposite that of the nearest pole in the control rod, the 
control rod was pulled toward the cap and away from the opening in the 
partition. When the electromagnet polarization was reversed, the nearest 
pole in the control rod was repelled, pushing the control rod away from 
the cap toward the opening in the partition. Thus, the control rod was 
externally repelled or attracted to occlude or open the opening in the 
partition, respectively. External control rod activation eliminated the 
risks of contamination. The cells in the culture chamber can be fed by 
engaging the electromagnetic to pull the control rod away from the opening 
in the partition, allowing mixing between the two chambers. Alternatively, 
early in the tissue construct development, the control rod is be pushed to 
occlude the opening and minimize mixing. Minimizing mixing would allow de 
novo growth factors to increase in concentration at a time when the tissue 
construct needs the highest concentration of growth factors. 
EXAMPLE 4 
In an alternate embodiment for automating media repletion, a dye 
incorporated into a matrix was placed in the tissue culture chamber that 
was sensitive to one or more environmental parameters. Said dye can be in 
solution or immobilized. As example, but limited to, phenol red (Sigma, 
St. Louis, Mo.) covalently bound to a matrix was utilized to monitor pH. A 
second example was ruthenium diimine complex (Sigma, St. Louis, Mo.) which 
was utilized to monitor dissolved oxygen. An external monitor that 
included an LED, photodiodes and a microcontroller, interrogated the 
dye/matrix to determine the absorbance change (phenol red) or fluorescent 
change (ruthenium diimine). Based on the calorimetric (or fluorometric) 
changes of the indicator dye, the electromagnet was energized to move the 
control rod and occlude the opening, or allow the media and tissue 
chambers to mix. External monitoring and control eliminated contamination 
risks. The entire culture roller bottle, control rod and indicator dye was 
disposable. A disposable automated tissue culture unit is especially 
suited to autologous tissue culture and suited to meeting federal 
regulatory requirements. 
It can be appreciated that the maintenance of a normal metabolic 
environment, through enhanced gas mass transfer and automation in the 
present invention, would improve the scientific capability for engineering 
tissues. Costs and FDA compliance are significantly improved by using a 
disposable culture roller bottle and standard rotating devices. 
EXAMPLE 5 
In an alternate embodiment for engineering sheets of tissue, normal human 
epidermal keratinocytes (Clonetics, Calif.) were placed in the tissue 
culture chamber with media (Clonetics, San Diego, Calif.). This method and 
product-by-process is suitable for cartilage constructs. The roller bottle 
was placed in a vertical position. The keratinocytes and other cells 
associated with the epidermal layer, were allowed to sediment to the 
bottom and attach to the wall opposite and parallel to the partition. 
After 4-7 days, depending on initial seeding densities, cells were 90% 
confluent. The roller bottle was then placed in a horizontal position and 
rotated, as previously described. Non-adherent cells were suspended in the 
tissue culture chamber by horizontal rotation. Suspended cells 
differentiated and adhered to the 90% confluent monolayer resulting in a 
sheet of tissue. The thickness of the developing sheet of tissue was 
governed by seeding density and the time maintained in culture. It will be 
appreciated by those of ordinary skill in the art that a variety of 
shapes, thickness and tissue phenotypes can be engineered. 
EXAMPLE 6 
In an alternate embodiment for proliferating human hepatocytes, normal 
human hepatocytes were cultured and expanded. A human liver enzymatic 
digest substantially consisting of normal human hepatocytes was obtained 
from Clonetics (San Diego, Calif.). The minor cellular elements in the 
digest were normal cellular constituents that comprise a normal human 
liver. Hepatocytes were suspended in culture media (Clonetics, San Diego, 
Calif.), then pipetted into the tissue culture chamber. The tissue culture 
chamber was filled with media to have zero headspace and the media chamber 
was filled with the same media. An ambient gas headspace filled the 
remaining 1/3 of the media chamber. The roller bottle was placed in a 
horizontal position and rotated as previously described. Typically, 
hepatocytes and other cellular constituents do not aggregate. However, in 
the present invention 3-dimensional tissue constructs developed. The 
constructs were suited to clinical therapies. It will be appreciated by 
one of ordinary skill in the art that the ability to grow and expand 
normal cells into tissue constructs has a variety of applications. 
EXAMPLE 7 
Frozen human islets are quick thawed and placed in standard culture media 
and 10% FBS. Several different medias are known in the art and can include 
CMRL 1066 media. Islets are cultured for the first 18-24 hours in a 
t-flask. The initial t-flask culture, helps to remove acinar that 
contaminate the culture and cellular debris from cells lost due to 
freezing/thawing. Cellular aggregates, predominately islets &gt;80%, are 
dislodged from the bottom of the t-flask by several blunt palm taps. 
Islets are then pelleted at 50.times.g for 5 minutes and 25% of the bottom 
volume is resuspended in the same culture media (except 5% FBS) that has 
been equilibrated to 80 mm Hg dO.sub.2, balance CO.sub.2. Islets are then 
placed in a 6 ml culture vessel of the present invention that is operated 
for 7-10 days in automated suspension mode, where environmental pH and 
oxygen are monitored and regulated. This step stabilizes the culture by 
improving the viability and allowing the cells that were in early 
apoptosis, as a result of pancreatic isolation procedures, to complete the 
programmed cell death and wash away. The 6 ml islet suspension is expected 
to be &gt;95% viable by day 7-10. After the first 7-10 days, the vessel is 
switched to static mode for 5-9 days, where the culture vessel is in a 
non-rotating vertical position. In static culture, islets will become 
disaggregated. This appears to the most gentle method for islet 
disaggragation. The vessel is then switched back to suspension mode, 
allowing the individual disaggregated cells to form cellular nucleation 
sites. Each nucleation site will both adhere to other cells that have a 
contact predilection for that subpopulation and, aggregate constituents 
will replicate/differentiate into 3-dimensional islet constructs. The 
first month is a fragile development period in any suspension culture. 
During the first month, extracellular formation is susceptible to 
disaggragation. Consequently, during the first month the 150.mu. diameter 
monitoring is by observation only. 
After the first month of culture, aggregates are more robust and better 
tolerate vessel handling. From month 2-3 the serum concentration is 
reduced from 5% to 2%. In this invention, once a construct has developed a 
stable autologous extracellular matrix, there is less of a need for serum 
and other growth factors. Furthermore, serum reduction measures will 
reduce costs and lessen the FDA screening requirements. Occasional 
representative samples are drawn with large bore pipette tips--small 
volumes so as not to deplete the developing culture. The number of cells 
in an islet aggregate is variable. Standard hemocytometer and coulter 
counter methods are accepted standards. In an alternate embodiment, the 
vessel will be monitored for biomass increases and aggregate size 
distribution, by laser light scatter. Briefly, as the density and size of 
the cellular aggregates change, the frequency of the laser beam disruption 
changes; as the vessel rotates through a laser beam that has been 
transformed to a line. Fast Fourier Transfer of the time domain data, 
collected from light scattering off the cellular aggregates, provides 
density and size distribution data that can be normalized to the first and 
last calibration samples. Integrating the intensity and size distribution 
data is proportional to the biomass. 
Samples taken from the 2-3 month maturation period are DTZ stained. It is 
anticipate that early during the maturation phase that DTZ staining will 
be very weak. Later, at the end of the maturation phase, islet constructs 
will be a pale pink. DTZ staining is better correlated with mature insulin 
packaging than extracellular insulin levels or islet function. After three 
months, representative samples are collected and tested for function. 
Islet construct functions are assessed by static glucose stimulation 
(SGS). Briefly, 100 islet equivalent (IEQ) islet constructs (define as 
150.mu. diameter islet construct) are washed and incubated in 50 mg % 
glucose for baseline determination. After baseline assessment, the same 
100 IEQs are incubated in 300 mg % glucose with 10 mM theophylline and 
spent media collected. The same batch is washed with 50 mg % glucose media 
and incubated in the baseline media, to obtain return to baseline 
measurements. Assuming return to baseline, a 2.times. insulin production 
over baseline is the minimum acceptance criteria for mature islet 
constructs. Alternately, islets derived from fetal or adult donors are 
utilized to create immature 150.mu. constructs. The 150.mu. constructs are 
transplanted into the host, maturing in the host to functional constructs. 
Once aggregation occurs, oxygen preconditioning begins. Early in the 
process oxygen tension is high to compensate for the loss of the vascular 
supply--when the islets were removed from the pancreas. As cell 
constituents of the islet re-aggregate to form a new islet construct, a 
preselection process begins. The new islet constructs become functional 
without a vascular supply. Over a 10-40 day period, the oxygen tensions 
are lowered to oxygen tensions expected to be found in the transplantation 
site. For unencapsulated islets, final oxygen tensions are approximately 
60 mm Hg. For encapsulated islets, oxygen tensions are 40-50 mm Hg. Both 
pH and oxygen are controlled, to maintain low oxygen tension concurrently 
with a physiological culture environment 
Islet encapsulation has been previously described. Islet constructs are 
washed once in calcium and magnesium free HBSS. The islets in HBSS are 
co-axially extruded with 1.5% alginate into a 0.5 cm 28 gauge tube for 
droplet generation. This reduces the alginate exposure time. The final 
alginate concentration is approximate 1% after co-extrusion. 
Alginate/islet drops, 1:1 islet/capsule, are generated into cell culture 
media with 5% FBS. The media with serum has sufficient free calcium to 
solidify droplets and enough protein to saturate alginate partitioning. 
In an alternate embodiment, islet aggregates are co-cultured with mammalian 
sertoli cells, resulting in sertoli/islet aggregates. The sertoli/islet 
constructs would be less immunogenic than other allogeneic or xenogeneic 
constructs. It is known in the art that sertoli cell produce FasL, an 
immunosuppressant factor. The construct is transplanted under the kidney 
capsule, or in an alternate embodiment, encapsulated and transplanted to 
the abdominal peritoneum. 
EXAMPLE 8 
In fertilizing and developing embryos, mammalian eggs and sperm are 
pipetted into the tissue culture chamber filled to a zero headspace with 
culture media suitable for fertilization and growth. The media chamber is 
filled with culture media to have a gas headspace as previously described. 
The vessel is tilted from a vertical position to a horizontal position and 
disposed to a means to horizontally rotate the vessel. Environmental 
oxygen and pH are monitored and controlled as previously described. Sperm 
bind to the eggs, penetrate and fertilize the eggs. Fertilized eggs in 
suspension undergo maturation, forming moruli, early blastocyts, then 
trophoblasts (when co-cultured). In addition to facilitating fertilization 
and maturation, this invention enables the growth of embryonic tissue free 
from immunological surveillance. It will be appreciated by those of 
ordinary skill in the art that this method and product-by-process has a 
variety of applications. Alternatively, endometrial tissue is co-cultured 
with the fertilized egg, later forming endometrial implantation constructs 
that support embryonic maturation. The endometrial elements will 
eventually form decidua with phenotypic and morphological attributes that 
characterize implantation. The process will continue until the construct 
becomes mass transfer limited and requires transplantation to a receptive 
uterus. It will be appreciated by those of ordinary skill in the art that 
the present invention mitigates the loss of eggs, sperm, and co-culture 
cell during the feeding. In a clinical in vitro fertilization setting, the 
loss of an egg could be catastrophic. 
EXAMPLE 9 
In proliferating cardiac myocytes, human cardiac myocytes with the cell 
constituents that support the primary architecture are obtained from 
Clonetics (San Diego, Calif.). The myocytes are washed in media 
(Clonetics, San Diego, Calif.), pelleted at 50.times.g and placed in the 
culture chamber. The culture chamber is filled to zero headspace with 
media, and the media chamber is 2/3 filled with media and 1/3 filled with 
ambient air. A cell monolayer will begin form at the bottom of the tissue 
culture chamber in 4-7 days. After an initial monolayer has formed, the 
vessel is tilted to a horizontal position and rotated. Rotation suspends 
the growing myocyte constructs while the wall monolayer serves as a feeder 
layer, providing growth factors and other support. Once the myocyte 
population is expanded to a biomass suitable for transplantation, the 
media is removed and constructs pelleted and washed in normal saline. 
Proliferated myocytes are injected into the site of myocardial damage. In 
an alternative embodiment, myocytes are selected from the group consisting 
of cardiac, skeletal, myocyte stem cells, and smooth muscle, and utilized 
for treatment of their origin. For example, proliferated skeletal muscle 
cells can be transplanted into skeletal muscle to overcome various 
muscular dystrophies. 
EXAMPLE 10 
In developing therapeutics for HIV, human lymphocytes are collected from 
the peripheral blood and/or lymph nodes. Explants or enzymatically 
dissociated cells are placed in to culture chamber and both culture and 
media chambers are filled with media as previously described. It is known 
in the art that lymphocytes traffic virus between lymph nodes. Suspension 
in the current invention would support trafficking between explants. Virus 
infected explants or native virus is placed in the culture chamber. Drugs 
are screened for activity by adding the drug, or combination of drugs, to 
the media at various stages in the inoculation. Samples are withdrawn and 
analyzed for viral load and cellular incorporation of the virus. 
Furthermore, the control rod can be used to regulate the timing and the 
amount of drug or virus that enters the culture chamber. Therapeutics or 
virus being placed in the media chamber and added to the culture chamber 
by controlling the occlusion of the opening in the partition. 
EXAMPLE 11 
In developing chimeric constructs, human cells and foreign human or 
non-human cells are co-cultured as previously described for normal 
tissues. The co-culture achieves a level of function that has a 
therapeutic benefit. In one of many instances know in the art, tissue 
macrophages are harmful to the developing construct. They can react to 
aberrant phenotypes or cellular metabolism, releasing cytokines and other 
products therein eliciting an inflammatory cascade. Inflammation can 
result in the rejection of a transplanted construct. Co-culturing of human 
islets or neuronal cells with similar cell types from an allogeneic or 
xenogeneic donor can activate both sets of tissue macrophages. Activation 
will eventually deplete the activated subpopulation, resulting in a 
surviving population substantially free from tissue macrophages. 
EXAMPLE 12 
In propagating virus, cellular constructs are cultured and matched with the 
virus that is know to grow in that cellular host. The predilection of 
certain viruses for specific tissues is known in the art, or specific gene 
therapies disposed to viral based vectors are utilized for gene therapy. 
The present invention facilitates the growth of both monolayer and 
3-dimensional tissue constructs, and mitigates the depletion of 
non-adherent cell phenotypes lost during feeding, resulting in a plethora 
of phenotypes that are not typically present in culture. Hence, tissue 
constructs are engineered to have autologous extracellular matrix, 
architecture, and phenotypes that are conducive to viral propagation. For 
example, mesenchymal cells obtain from human donor proximal intestinal 
tissue (or neuronal cells for neurotrophic viruses) are placed in the 
tissue culture chamber as previously described. The cells are cultured for 
10-14 days to allow for adaptation, autologous extracellular matrix 
formation, and for the expression of phenotypes that are unique to the 
novel culture vessel and environment of this invention. After 10-14 days, 
10 .mu.l of virus obtained from Centers for Disease Control is add to the 
tissue culture chamber. Following two viral doubling periods, samples are 
collected and analyzed for intracellular virus and free virus. Lytic 
viruses that tend to deplete standard cultures of their cellular host, 
will continue to propagate for longer periods due to the cellular 
regenerative capacity implicit in the tissue constructs of this novel 
culture vessel and environment. 
In an alternate embodiment, the viral vector contains specific sequences 
for the production of recombinant protein. In the present invention, a 
greater level of recombinant production is achieved, owing the improvement 
to the regenerative capacity of the growing constructs. 
In an alternate embodiment, viral vectors include knock-out sequences for 
incorporation into the genome. Cellular knock-outs proliferate in the 
present invention can be utilized in basic research and transplantation. 
EXAMPLE 13 
In propagating bone marrow, neonatal cord blood or bone marrow collections 
are cultured in the tissue culture chamber. The stem cells from the cord 
blood or bone marrow along with the more mature cell constituents, are 
cultured in suitable media as a monolayer for 2-3 days. During the first 
few days of monolayer culture, the cells will gently disaggregate and form 
a monolayer with patches of multicellular constructs. The multicellular 
patches and monolayer will later become the stromal feeder layer. The 
culture vessel is then horizontally tilted and rotated. Non-adherent stem 
cells will detach from the monolayer and stromal patches and become 
suspended. As the suspended cell proliferate and differentiate, new 
adherent phenotypes will develop and adhere to the feeder layer. After 30 
days, enough marrow will have been propagated to seed additional vessels, 
and eventually, depending on the weight of the recipient, enough for 
transplantation. 
EXAMPLE 14 
In producing a validated product-by process, hyaline cartilage obtained by 
surgical means from the knee is enzymatically dissociated and placed in 
the tissue culture chamber. Cartilage is cultured as a monolayer for 2-3 
days therein forming a monolayer with patches of multicellular constructs. 
The vessel is then rotated and pH and oxygen monitored and controlled for 
22 days. Oxygen is maintained at low oxygen tension, approximately 40 mm 
Hg. The same process is reproduced several times to establish a typical pH 
and oxygen utilization profile with the required degrees of freedom to 
detect statistical outliers. Once a standard profile has been established, 
processes falling three standard deviations from the normal profile, are 
rejected. Aberrancies caused by atypical starting material, contamination, 
or quality control deficiencies, are identified and the autologous tissue 
lot rejected. Hence, the product-by-process is the autologous construct 
that matches the standard profile for oxygen and pH changes. In many 
instances, it is the standard of practice to qualify tissue after it has 
been cultured or transplanted. Real time monitoring and control of oxygen 
and pH changes improves safety by identify contaminated tissue or atypical 
tissue prior to transplantation, and improves reproducibility. 
EXAMPLE 15 
The sensor platform consists of: LEDs for interrogating sensor dyes or 
dye/matrix, a low drift photosensor subassembly (non-linearity .+-.0.2% 
F.S., supply sensitivity 0.5%/V, temp. coef. .+-.100 ppm/.degree.C., 20 
bits resolution), and requires 5 V @ 2 mA average), the MC68HC705P9 
microcontroller, and an interface (direct, IR or USB depending on the 
application). Alternatively, a sampling 16-bit analog to digital converter 
can be used. Estimated MTBF for the platform is approximately 50,000 hours 
(the individual components have MTBF typically &gt;100,000 hours). The sensor 
platform drifts less than 0.5% per year, consumes, on average, 10 .mu.A 
power @ 5 V, peak power consumption is 20 mA @ 5 V, and can collect 
approximately 4,000 measurements powered from a 9 V alkaline battery. The 
sensor platform is a generic platform; to change from pH to dO.sub.2 to 
glucose, only the solid matrix containing the sensing dye and the 
appropriate wavelength LEDs are changed, for dO.sub.2 a filter is added. 
The matrix consists of a porous material that is sterilized and placed in 
the cell culture. An example of dye/matrix material includes: silicone for 
the ruthenium diimine complex and fibrous cellulose for the covalently 
coupled phenol red. 
It will be apparent to those skilled in the art that various changes may be 
made in the invention without departing from the spirit and scope thereof, 
and therefore the invention is not limited by that which is disclosed in 
the drawings and specifications but only as indicated in the appended 
claims.