U.S. patent application number 10/166732 was filed with the patent office on 2002-11-21 for normothermic, hypothermic and cryopreservation maintenance and storage of cells, tissues and organs in gel-based media.
This patent application is currently assigned to BioLife Solutions, Inc.. Invention is credited to Baust, John G., Baust, John M., Van Buskirk, Robert.
Application Number | 20020172934 10/166732 |
Document ID | / |
Family ID | 26871776 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020172934 |
Kind Code |
A1 |
Baust, John M. ; et
al. |
November 21, 2002 |
Normothermic, hypothermic and cryopreservation maintenance and
storage of cells, tissues and organs in gel-based media
Abstract
Gel-based medium compositions and a method of use thereof in
normothermic, hypothermic or cryopreservative storage and transport
of cell samples are described. These gel-based compositions contain
a cell maintenance and preservation medium together with a gelling
agent. Such gel-based medium compositions protect various cell
samples, such as animal or plant organs, tissues and cells, from
the mechanical, physiological and biochemical stresses inherently
associated with liquid preservation techniques.
Inventors: |
Baust, John M.; (Vestal,
NY) ; Van Buskirk, Robert; (Apalachin, NY) ;
Baust, John G.; (Candor, NY) |
Correspondence
Address: |
PILLSBURY WINTHROP, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
BioLife Solutions, Inc.
|
Family ID: |
26871776 |
Appl. No.: |
10/166732 |
Filed: |
June 12, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10166732 |
Jun 12, 2002 |
|
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|
09757694 |
Jan 11, 2001 |
|
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60176009 |
Jan 14, 2000 |
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Current U.S.
Class: |
435/2 ;
435/383 |
Current CPC
Class: |
A01N 1/0231 20130101;
A01N 1/02 20130101; A01N 1/0226 20130101; A01N 1/0205 20130101 |
Class at
Publication: |
435/2 ;
435/383 |
International
Class: |
A01N 001/02; C12N
005/02 |
Claims
What is claimed is:
1. Gel-based medium compositions for transport or storage of cell
samples, the gel-based compositions comprising at least one cell
maintenance and preservation medium and at least one gelling
agent.
2. The gel-based medium compositions according to claim 1, wherein
transport or storage of cells occurs at a temperature selected from
the group consisting of normothermic, hypothermic and
cryopreservative temperatures.
3. The gel-based medium compositions according to claim 2, wherein
transport or storage of cell samples occurs at a temperature
ranging from about -196.degree. C. to about 37.degree. C.
4. The gel-based medium compositions according to claim 1, wherein
the cell samples are obtained from a source selected from the group
consisting of plants, animals, fungi, and microbes.
5. The gel-based medium compositions according to claim 4, wherein
the cell samples are selected from the group consisting of organs,
tissues, cell monolayers and single cells.
6. The gel-based medium compositions according to claim 5, wherein
the cell samples are obtained from humans.
7. The gel-based medium compositions according to claim 1, wherein
the at least one cell maintenance and preservation medium is
liquid.
8. The gel-based medium compositions according to claim 1, wherein
the at least one gelling agent is a member selected from the group
consisting of gelatin, carrageenan, agarose, collagen and
plant-based gelling agents.
9. A gel-based medium composition for transport or storage of cell
samples, the composition comprising: (a) one or more electrolytes
selected from the group consisting of potassium ions at a
concentration ranging from about 10-145 mM, sodium ions ranging
from about 10-120 mM, magnesium ions ranging from about 0.1-10 mM,
and calcium ions ranging from about 0.01-1.0 mM; (b) a
macromolecular oncotic agent having a size sufficiently large to
limit escape from the circulation system and effective to maintain
oncotic pressure equivalent to that of blood plasma and selected
from the group consisting of human serum albumin, polysaccharide
and colloidal starch; (c) a biological pH buffer effective under
physiological and hypothermic conditions; (d) a nutritive effective
amount of at least one simple sugar; (e) an impermeant and hydroxyl
radical scavenging effective amount of mannitol; (f) an impermeant
anion impermeable to cell membranes and effective to counteract
cell swelling during cold exposure, said impermeant ion being at
least one member selected from the group consisting of
lactobionate, gluconate, citrate and glycerophosphate-like
compounds; (g) a substrate effective for the regeneration of ATP,
said substrate being at least one member selected from the group
consisting of adenosine, fructose, ribose and adenine; (h) at least
one agent which regulates cellular levels of free radicals; and (i)
at least one gelling agent.
10. The gel-based medium composition according to claim 9, wherein
the gelling agent is a member selected from the group consisting of
gelatin, carrageenan, agarose, collagen and plant-based gelling
agents.
11. The gel-based medium composition according to claim 9, wherein
the transport or storage of cell samples occurs at a temperature
ranging from about -196.degree. C. to about 37.degree. C.
12. The gel-based medium composition according to claim 9, wherein
the cell samples are obtained from a source selected from the group
consisting of plants, animals, fungi, and microbes.
13. The gel-based medium composition according to claim 12, wherein
the source of the cell samples is a fetal, neonatal, juvenile or
adult animal.
14. The gel-based medium composition according to claim 13, wherein
the cell samples are obtained from humans.
15. A method of storing cell samples in a gel-based medium
composition, the method comprising: warming a gel-based medium
composition to melt a gelling agent contained therein; suspending
cell samples in a cell preservation solution without a gelling
agent; mixing the suspended cell samples with the warmed gel-based
medium composition; cooling the cell samples to solidify the
gelling agent; and transferring the cooled cell samples to a
desired storage temperature.
16. The method according to claim 15 further comprising optionally
transporting the cooled cell samples in a gelled state.
17. The method according to claim 16, wherein the transport or
storage of cell samples occurs at a temperature ranging from about
-196.degree. C. to about 37.degree. C.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to gel-based compositions for
normothermic, hypothermic and cryopreservative transport or storage
of plant tissues or cells and animal organs, tissues or cells, the
gel-based compositions comprising a cell maintenance and
preservation medium and a gelling agent.
[0003] 2. Description of Related Art
[0004] Today, limited normothermic, hypothermic and
cryopreservative maintenance and storage of plant or mammalian
cells, tissues and organs (biologics) is accomplished in liquid
media. Success is limited, in part, due to damage that occurs
during shipment (transport), most often associated with mechanical
trauma.
[0005] Preservation and transport (herein referred to as
preservation) of biologics (cells, tissues, and organs) has
traditionally been achieved through "suspension" in a liquid
preservation medium. These media include, but are not limited to,
simple saline solution, cell culture media, and preservation
solutions such as University of Wisconsin (UW) solution
(VIASPAN.RTM.), EURO-COLLINS.RTM., and HYPOTHERMOSOL.RTM. (Bio Life
Solutions, Inc., Ewing, N.J., USA). Inherent in this liquid
preservation approach is that the liquid environment confers no
physical support network for the biologic during preservation and
transport. Due to this lack of physical support upon preservation,
biologics are exposed to numerous physical stresses during storage
and shipment. These stresses include, but are not limited to,
sedimentation, mechanical "jarring", compaction in a liquid column,
shaking, vibration, shearing forces, ice damage, and the like. As a
result of these mechanical stresses plus additional biochemical
stresses inherently associated with biologic preservation in
liquid, a significant level of cellular death is initiated during
and following the preservation interval. Consequently, failure of
the biologic ensues due to this preservation-initiated cell
death.
SUMMARY OF THE INVENTION
[0006] The invention relates to gel-based medium compositions for
normothermic, hypothermic or cryopreservative transport and/or
storage of plant tissues or cells and animal organs, tissues or
cells, the gel-based compositions comprising a cell maintenance and
preservation medium and a gelling agent. In particular, mammalian
samples, such as human and animal organs, tissues and cells, may be
preserved in the inventive gel-based media compositions.
[0007] In a preferred embodiment, the cell maintenance and
preservation medium is liquid.
[0008] In another embodiment, the gel-based medium compositions
comprise:
[0009] (a) one or more electrolytes selected from the group
consisting of potassium ions at a concentration ranging from about
10-145 mM, sodium ions ranging from about 10-120 mM, magnesium ions
ranging from about 0.1-10 mM, and calcium ions ranging from about
0.01-1.0 mM;
[0010] (b) a macromolecular oncotic agent having a size
sufficiently large to limit escape from the circulation system and
effective to maintain oncotic pressure equivalent to that of blood
plasma and selected from the group consisting of human serum
albumin, polysaccharide and colloidal starch;
[0011] (c) a biological pH buffer effective under physiological and
hypothermic conditions;
[0012] (d) a nutritive effective amount of at least one simple
sugar;
[0013] (e) an impermeant and hydroxyl radical scavenging effective
amount of mannitol;
[0014] (f) an impermeant anion impermeable to cell membranes and
effective to counteract cell swelling during cold exposure, said
impermeant ion being at least one member selected from the group
consisting of lactobionate, gluconate, citrate and
glycerophosphate-like compounds;
[0015] (g) a substrate effective for the regeneration of ATP, said
substrate being at least one member selected from the group
consisting of adenosine, fructose, ribose and adenine;
[0016] (h) at least one agent which regulates cellular levels of
free radicals; and
[0017] (i) at least one gelling agent.
[0018] Yet another embodiment of the invention is a method of
storing cell samples in a gel-based medium composition, the method
comprising:
[0019] warming a gel-based medium composition to melt a gelling
agent contained therein;
[0020] suspending cell samples in a cell preservation solution
without a gelling agent;
[0021] mixing the suspended cell samples with the warmed gel-based
medium composition;
[0022] cooling the cell samples to a chilled or frozen state,
thereby solidify the gelling agent; and
[0023] transferring the chilled or frozen cell samples to a desired
storage temperature.
[0024] Optionally, the chilled or frozen cell samples may be
transported in the gelled state.
BRIEF DESCRIPTION OF THE FIGURES
[0025] The invention is further illustrated in the Figures in
which:
[0026] FIG. 1 graphically depicts post-storage recovery of MDCK
(Madin Darby Canine Kidney) cells stored as monolayers in culture
plates at 4.degree. C. for 24 hours in liquid maintenance and
preservation solutions (controls) (media, HTS, & HTS FRS) and
in liquid maintenance and preservation solutions as gel
formulations (HTS Gel & HTS FRS Gel);
[0027] FIG. 2 graphically depicts post-storage recovery of MDCK
cells stored as monolayers in culture plates at 4.degree. C. for 3
days in liquid maintenance and preservation solutions (controls)
(media, HTS, & HTS FRS) and in liquid maintenance and
preservation solutions as gel formulations (HTS Gel & HTS FRS
Gel).;
[0028] FIG. 3 graphically depicts post-storage recovery of MDCK
cells stored in suspension in culture plates at 4.degree. C. for 24
hours in liquid maintenance and preservation solutions (controls)
(media, HTS, & HTS FRS) and in liquid maintenance and
preservation solutions as gel formulations (HTS Gel & HTS FRS
Gel);
[0029] FIG. 4 graphically depicts post-storage recovery of MDCK
cells stored in suspension in culture plates at 4.degree. C. for 3
days in liquid maintenance and preservation solutions (controls)
(media, HTS, & HTS FRS) and in liquid maintenance and
preservation solutions as gel formulations (HTS Gel & HTS FRS
Gel);
[0030] FIG. 5 graphically depicts post-storage recovery of human
pancreatic Islets of Langerhans micro-organs stored in suspension
in culture plates at 22.degree. C. for 1 day in maintenance and
preservation solutions as gel formulations;
[0031] FIG. 6 graphically depicts post-storage recovery of human
pancreatic Islets of Langerhans micro-organs stored in suspension
in culture plates at 22.degree. C. for 3 days in maintenance and
preservation solutions as gel formulations;
[0032] FIG. 7 graphically depicts post-storage recovery of human
pancreatic Islets of Langerhans micro-organs stored in suspension
in culture plates at 8.degree. C for 1 day in maintenance and
preservation solutions as gel formulations; and
[0033] FIG. 8 graphically depicts post-storage recovery of human
pancreatic Islets of Langerhans stored in suspension in culture
plates at 8.degree. C. for 3 days in maintenance and preservation
solutions as gel formulations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0034] To reduce and even eliminate damage associated with
mechanical factors arising from cell storage and transport,
gel-based maintenance and preservation media have been developed to
sustain biologics in supporting environments of various viscosity.
The gel-based media are rigid or highly viscous at temperatures
equal to or lower than 28.degree. C. but liquid at 37.degree. C.
Liquid maintenance and preservation media (i.e.,
HYPOTHERMOSOL.RTM., VIASPAN.RTM. (also called University of
Wisconsin solution), EUROCOLLINS.RTM., CARDISOL.RTM., UNISOL.RTM.,
tissue culture media, etc.) are converted to a gel of appropriate
viscosity necessary to match cell, tissue or organ type with either
animal or plant derived gelling agents (1-2% by volume). Suitable
combinations of such liquid maintenance and preservation solutions
may be used if desired. A preferred organ preservation solution is
HYPOTHERMOSOL.RTM., which is composed of:
[0035] (a) one or more electrolytes selected from the group
consisting of potassium ions at a concentration ranging from about
10-145 mM, sodium ions ranging from about 10-120 mM, magnesium ions
ranging from about 0.1-10 mM, and calcium ions ranging from about
0.01-1.0 mM;
[0036] (b) a macromolecular oncotic agent having a size
sufficiently large to limit escape from the circulation system and
effective to maintain oncotic pressure equivalent to that of blood
plasma and selected from the group consisting of human serum
albumin, polysaccharide and colloidal starch;
[0037] (c) a biological pH buffer effective under physiological and
hypothermic conditions;
[0038] (d) a nutritive effective amount of at least one simple
sugar;
[0039] (e) an impermeant and hydroxyl radical scavenging effective
amount of mannitol;
[0040] (f) an impermeant anion impermeable to cell membranes and
effective to counteract cell swelling during cold exposure, said
impermeant ion being at least one member selected from the group
consisting of lactobionate, gluconate, citrate and
glycerophosphate-like compounds;
[0041] (g) a substrate effective for the regeneration of ATP, said
substrate being at least one member selected from the group
consisting of adenosine, fructose, ribose and adenine; and
[0042] (h) at least one agent which regulates cellular levels of
free radicals.
[0043] By adjusting the formulation of 1) the carrier medium, in
particular, a cell maintenance and preservation solution, 2)
gel-type and 3) gel concentration, an appropriate long-term (up to
two weeks) shipping environment has been produced, and is
commercially known as GELSTOR.TM. (Biolife Solutions, Inc., Ewing,
N.J., USA). Procedurally, a biologic of choice is exposed to
liquefied gel at 37.degree. C., and thereafter cooled to a desired
preservation temperature for maintenance and shipment. Upon arrival
or point-of-use, the gel is warmed to 37.degree. C. whereupon it
liquefies, and may be decanted. The biologic may then be rinsed
with an appropriate medium and is ready for use.
[0044] The gel-based preservation and maintenance technology
disclosed herein has been developed to provide a semi-solid
preservation matrix that facilitates protection of various cells
from mechanical stresses associated with biologic preservation and
transport. In addition to physical protection, gel-based
preservation media provide physiological and biochemical protection
during the storage and transport of cells, organs and tissues.
[0045] A variety of animal and plant cells may be stored and
transported in gel-based media, including human cells and animals
cells from numerous tissue sources, including tumor cells, cells
from the liver, kidney, central nervous system, epidermal
keratinocytes, endothelial cells, stem cells, white blood cells,
fibroblasts, pancreatic islet cells, cardiac and skeletal muscle
cells, sperm, egg, satellite cells and the like. Fetal, neonatal,
juvenile and adult cells, tissues and organs all benefit from the
protective aspects of the inventive gel-based carrier media. In
particular, cells from younger sources are highly sensitive to the
mechanical stresses of preservation and transport, and thus are
ideal candidates for the mechanically protective aspects of a
gel-based preservation medium. Conversely, cells from older sources
are highly susceptible to damage from oxidative stresses incurred
during preservation and transport, meaning these cells particularly
benefit from the physiological and biochemical protection afforded
by gel-based preservation media.
[0046] Cell lines and tissues from which cell lines are to be
developed are suitable for storage and/or preservation in gel-based
media. Likewise, organs and tissues from donor animals destined for
transplantation into other animals, including humans, will have
greater viability upon storage and transport in gel-based media.
Plant cells and tissues, such as those from transformed or
transgenic sources may be stored and/or transported in gel-based
media. Likewise, tissue grafts of plants are suitable samples for
preservation and, if desired, transport in a gel-based medium.
Microbial and fungal cells would also benefit form storage and
transport in a gel-based preservation media. For example, microbial
systems used in environmental remediation, such as
bacterially-mediated oil degradation or sewage treatment, may be
successfully transported to a point of use in a gel-based
preservation medium.
[0047] Traditionally, preservation and transport of single cells
has relied on suspension of cells in a liquid-based medium. Such
liquid-based preservation regimes result in the settling of cells
to the bottom of the preservation container. This settling causes
an inhomogeneous distribution of the cells in the medium resulting
inefficient exposure of the cells to the preservation solution, as
well as the exposure of the cells to mechanical stresses of
"jarring and shearing" experienced during shipment. Accordingly,
gelled preservation solutions permit maintenance of cells in
suspension during periods of preservation. This maintenance of
cellular suspension eliminates cell sedimentation and reduces
mechanical stresses experienced by the cells subjected to storage
and/or transport conditions. Additionally, the composition of the
inventive gelled preservation medium is designed to provide a
biochemical environment beneficial to cellular preservation. This
protective environment is facilitated through the incorporation of
an organ preservation solution as the principal diluent in which
the gellation component of the medium is mixed.
[0048] Cellular monolayers are sheets of cells one to a few cell
layers thick that are grown on an inert matrices in vitro. These
monolayers are typically preserved in liquid based preservation
mediums. Due to preservation in liquid-based media, monolayer
separation from the growth matrix occurs during preservation. This
separation results in the dissembling of the system and ultimately
failure caused by the preservation process. Preservation of
cellular monolayers using the inventive gelled medium prevents
cell-matrix separation during the preservation interval.
Additionally, gelled media provide the same preservation benefits
to cellular monolayers as is conferred upon cellular
suspensions.
[0049] Biologic tissues are multi-layered cellular constructs that
interact to perform a particular function. The complexity of
tissues ranges from engineered tissue constructs consisting of a
single cell type (i.e. EPIDERM.TM. MatTech, Ashland, Mass., USA) to
human skin grafts, to "micro-organs" (i.e. pancreatic islets), to
whole human organs (i.e. kidneys, livers, etc.). Preservation of
these cell systems, as with single cells and monolayers, is
typically performed utilizing liquid preservation media. Due to the
complex nature of tissues, the shortcomings of liquid-based
preservation technologies include those associated with cell and
monolayer preservation, along with the inability to maintain the
more intricate cell-cell interactions that are stressed during
periods of preservation.
[0050] The inventive gelled preservation media enables preservation
of tissue in a semi-solid preservation matrix keeping the tissue
structurally intact while affording the same protective benefits to
the tissue as is conferred to individual cells and cell monolayers.
Specifically, the reduction in the external mechanical shipping
forces experienced by the tissues shipped in the inventive gel
medium markedly improves tissue viability following preservation.
For example, pancreatic islet cells preserved in a medium
comprising gelatin and HYPOTHERMOSOL.RTM. were afforded significant
improvement of islet integrity and functionality following
preservation.
[0051] Types of gellation material suitable for use in a gelled
cell preservation medium include gelatin, carrageenan, agarose,
collagen, plant-based gelling agents, combinations thereof and the
like. Plant-based gelling agents include gels produced from the
roots, stems, tubers, fruit and seeds of plants.
[0052] The invention is further described in the following
non-limiting examples.
EXAMPLES
Example 1
Gel-Based Medium Preparation
[0053] Calculations are first performed to determine the necessary
volume of gel-based medium for a given storage or transport need.
The desired gel concentration must be established. Typically, a
standard concentration of 2% is used, although this concentration
may vary depending upon the characteristics of the biologic being
preserved. A stock solution of gel-based medium was then prepared
(standard=14%) in a sterile environment. The volume of stock
solution needed was determined and the mass of the appropriate
amount of gelatin powder (Sigma Chemicals, St. Louis, Mo., USA) was
ascertained. The appropriate volume of organ preservation solution
(HYPOTHERMOSOL.RTM., or "HTS") was measured and combined via
agitation with the gelatin powder. In this instance, the stock
solution was mixed by swirling the container having the HTS and
gelatin, although any suitable means of agitation may be employed.
The stock solution of gel-based medium was then warmed in a
40.degree. C. water bath for 15-20 min with repeated swirling
solution (once per minute) to dissolve the gelatin. A 2% solution
of gel-based medium was then prepared from this stock solution. The
desired volume depended upon the quantity of cells to be preserved.
For example, for 100 ml of the 2% solution of gel-based medium, 14
ml of 14% stock solution of gel-based medium was combined with 86
ml of an HTS-Free Radical System (FRS) solution. Aliquots of this
2% solution of gel-based medium were dispensed into 15 ml
centrifuge tubes at 10 ml per tube, which were stored at 4.degree.
C. until used.
Example 2
Gel-Based Medium Storage Protocol
[0054] Aliquots of previously prepared 2% gel-based medium were
removed from 4.degree. C. storage and placed into a 37.degree. C.
water bath for 15 minutes to melt the gelatin contained therein.
While the gel-based medium was warming, samples destined for
preservation therein were prepared in a sterile environment. The
desired number of cells to be preserved were transferred into a
clean centrifuge tube and were gently centrifuged to pellet cells.
Typically, centrifugation at 500.times. g for 6 min is sufficient
to generate a cell pellet from which a supernatant can be decanted.
Pelleted cells were then suspended in 0.5-1.0 ml of HTS-FRS
solution without gelatin, which is an appropriate volume for
preservation in 2% gel-based HTS-FRS medium. The warmed 2%
gel-based medium, now in solution form, was removed from the water
bath and the suspended cells were pipetted into the warmed medium
in a sterile cell culture environment. After tightly securing lids
onto the sample storage tubes, the tubes were immediately placed
into an ice-water bath for 5 minutes to allow for rapid
solidification of the gel-based medium solution. A 2% gel-based
medium solution will solidify around room temperature in
approximately 30 min, and chilling was used to accelerate the
solidification. Chilled sample tubes were then transferred to a
desired storage temperature.
[0055] Samples prepared in this manner can be stored for any
desired time period at appropriate temperatures. For example, cells
may be stored for less than 24 hours or for as long as about three
days. Storage temperatures can range, for example, from about
-196.degree. C. to about +30.degree. C.
[0056] Temperatures are "normothermic" in a range between
31.degree. C.-37.degree. C. Temperatures are considered
"hypothermic" in a range between 0.degree. C. and 30.degree. C.
Cryopreservation generally occurs at temperature below 0.degree.
C., and may be effected using a combination of a gel-based media
with cryopreservants.
[0057] Sample tubes are removed from storage when the cells
contained therein are to be used. Accordingly, the sample tubes is
this instance were placed in a 37.degree. C. water bath for 6-8
minutes to melt the gelled medium. However, the gelling agent can
be formulated into a preservation medium such melting can occur at
any desired temperature above or below 37.degree. C. While samples
were thawing, tubes were inverted every 30 seconds to maintain
uniform temperature throughout the samples. Once the gelled medium
containing sample cells melted, samples were immediately removed
from the water bath. Cell samples were then gently centrifuged to
form pellets, typically at 37.degree. C. at 500.times. g for 6 min.
Cell sensitivity to heat stress will delimit tolerable temperature
ranges for gel melting for a given biologic. Centrifugation can be
performed at room temperature, but the gel-based medium solution
may partially resolidify at this temperature. Resolidification
causes uncontrolled gel concentration within the cell pellet during
centrifugation. For this reason centrifugation at temperatures
between 30.degree. C.-37.degree. C. is preferred.
[0058] After centrifugation, the gel-based medium supernatant was
decanted from the cell pellet, which was then suspended in 12 ml of
an appropriate cell culture medium at 37.degree. C. to wash
residual gel solution from the cells. The samples were then gently
centrifuged to pellet cells at 37.degree. C., typically at
500.times. g for 6 min. The supernatant was decanted from the
pelleted cells prior to resuspension to a desired cell density in
an appropriate volume of cell culture media at 37.degree. C.
Suspended cells were then be transferred to cell culturing vessels
at the density desired for growth.
[0059] While a preferred form of the invention has been herein
described, a skilled artisan would understand that the present
disclosure is by way of example and that variations are possible
without departing from the subject matter described within the
scope of the entire invention disclosed herein. All published
materials cited herein are hereby incorporated in their entirety by
reference.
* * * * *