U.S. patent application number 11/228388 was filed with the patent office on 2006-03-23 for method of cryopreserving cells.
Invention is credited to Janet Anne Wade Elliott, Locksley Earl McGann, Lisa Ula Ross-Rodriguez.
Application Number | 20060063141 11/228388 |
Document ID | / |
Family ID | 36074477 |
Filed Date | 2006-03-23 |
United States Patent
Application |
20060063141 |
Kind Code |
A1 |
McGann; Locksley Earl ; et
al. |
March 23, 2006 |
Method of cryopreserving cells
Abstract
A non-linear cooling cryopreservation method for improving
cryopreservation protocols for cells that involves producing a
simulation of cellular responses to a range of cooling parameters;
determining optimal cooling parameters required to minimize
cryoinjury to the cells using simulation of cellular responses and
experimental results; and incorporating optimal parameters into the
protocol. The simulation is based on mathematical models of
cellular parameters. A non-linear cooling cryopreservation protocol
for cryopreserving stem cells is also disclosed that does not
require cryoprotectants.
Inventors: |
McGann; Locksley Earl;
(Spruce Grove, CA) ; Elliott; Janet Anne Wade;
(Edmonton, CA) ; Ross-Rodriguez; Lisa Ula; (St.
Albert, CA) |
Correspondence
Address: |
MCCARTHY TETRAULT LLP
BOX 48, SUITE 4700,
66WELLINGTON STREET WEST
TORONTO
ON
M5K 1E6
CA
|
Family ID: |
36074477 |
Appl. No.: |
11/228388 |
Filed: |
September 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60611391 |
Sep 18, 2004 |
|
|
|
Current U.S.
Class: |
435/1.3 ;
702/19 |
Current CPC
Class: |
A01N 1/0284 20130101;
A01N 1/02 20130101 |
Class at
Publication: |
435/001.3 ;
702/019 |
International
Class: |
A01N 1/02 20060101
A01N001/02; G06F 19/00 20060101 G06F019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 17, 2004 |
CA |
2,482,045 |
Claims
1. A method for non-linear cooling cryopreservation of cells
comprising determining an optimal cooling profile for maximum
recovery of the cells and applying the cooling profile to the
cells.
2. The method for non-linear cooling cryopreservation of claim 1,
wherein any optimal cooling profile is determined in part or in
whole using a simulation of cellular responses to cooling
parameters.
3. The non-linear cooling cryopreservation method of claim 2,
wherein the cooling parameters comprise temperature, duration of
temperature exposure, amount of supercooling, cooling rate and the
cryoprotectant and concentration thereof.
4. The non-linear cooling cryopreservation method of claim 2,
wherein the cellular responses are selected from the group of
responses consisting of but not limited to: a) the maximum degree
of intracellular supercooling over the course of the cooling
protocol as a predictor of cryoinjury due to intracellular
freezing, b) the maximum intracellular potassium chloride
concentration as a predictor of cryoinjury due to exposure to the
concentrated solutes.
5. The non-linear cooling cryopreservation method of claim 4
wherein the simulation of cellular responses is determined in part
or in whole from mathematical models comprising: osmotic transport
properties, phase diagrams, and compostition and thermodynamic
parameters for the intra- and extra-cellular solutions for a
particular cell type.
6. A non-linear cooling cryopreservation protocol according to
claim 1, wherein the cryopreservation protocol comprises cooling
the cells to a first temperature holding for a first period of
time, then cooling the cells to a storage temperature for a second
period of time.
7. A non-linear cooling cryopreservation method according to claim
6, wherein the cryopreservation protocol is used with cells stored
without cryoprotectants.
8. A non-linear cooling cryopreservation method of claim 7 wherein
the cryopreservation method is used with cells stored with a
pentrating cryoprotectant.
9. A non-linear cooling cryopreservation method of claim 8 wherein
the cryopreservation method is used with cells stored with
infusible permeating cryoprotectant.
10. A non-linear cooling cryopreservation protocol of claim 7
wherein the cryopreservation method is used with cells stored with
a non-pentrating cryoprotectant.
11. A non-linear cooling cryopreservation method according to claim
8, wherein the non-permeating cryoprotectant comprises but is not
limited to sugars, starches, serum, proteins, or plasma.
12. A non-linear cooling cryopreservation method according to claim
1, wherein the cells are selected from the group consisting of:
stem cells, progenitor cells, red and white blood cells, sperm
cells, oocytes, ova, cells for research or transplant purposes,
cellular materials derived from tissues and organs, pancreatic
islet cells, chondrocytes, cells of neural origin, cells of hepatic
origin, cells of ophthalmolic origin, cells of orthopedic origin,
cells from connective tissues, and cells of reproductive origin,
and cells of cardiac origin.
13. A non-linear cooling cryopreservation method according to claim
12, wherein the stem cells comprise peripheral blood stem cells,
human umbilical cord blood stem cells and stem cells derived from
tissues and solid organs or other sources, including fetal and or
embryonic sources.
14. A method for optimizing a method for cryopreservation of cells,
the method comprising: a) producing a simulation of cellular
responses to a range of cooling parameters; b) using the
information derived from the simulation of cellular responses,
determining an optimal cooling profile; c) using the optimal
cooling profile into the cryopreservation method; and optionally d)
further optimizing protocol with results from biological
experiments using the simulated optimal cellular responses.
15. A method according to claim 14, wherein the cooling parameters
comprise cell temperatures, temperature exposure duration periods,
cooling rates, amounts of supercooling, and nature and
concentration of cryoprotectants.
16. A method according to aspect 14, wherein the simulation of
cellular responses comprises calculating cellular responses from
mathematical models using osmotic transport properties, phase
diagrams, and compostition and thermodynamic parameters for the
intra- and extra-cellular solutions for a particular cell type.
17. A non-linear cooling cryopreservation method optimized
according to claim 14.
18. A method for optimizing a cryopreservation protocol for
cryopreserving cells wherein the cells are cooled to a first hold
temperature for a first period of time, then cooled to a second
storage temperature at which the cells are stored for a second
period of time before the cells are thawed, the method comprising:
a) producing a simulation of cellular responses to a range of
cooling parameters based on information derived from a simulation
of cellular responses to determine an optimal range of first hold
temperature, first range of time periods, and amount of
intracellular supercooling and/or amount of intracellular solute
concentration required to minimize cryoinjury to the cells; and/or
b) incorporating the optimal first hold temperature, optimal first
period of time, and optimal amount of intracellular supercooling
into the non-linear cooling cryopreservation method.
19. A method according to claim 18, wherein the cooling parameters
comprise cell temperatures, temperature exposure duration periods,
cooling rates, amount of supercooling, and nature and amounts of
cryoprotectants.
20. A method according to claim 18, wherein the simulation of
cellular responses comprises calculating cellular responses from
mathematical models using osmotic transport properties, phase
diagrams, and composition and thermodynamic parameters for the
intra- and extra-cellular solutions for a particular cell type.
21. A method for the cryopreservation of stem cells, comprising: a)
cooling the stem cells to a first temperature holding at that
temperature for a first period of time; and b) cooling the stem
cells to a second temperature for storing the cells for a second
period of time.
22. The method of claim 21 wherein the first temperature is between
-5.degree. C. and -30.degree. C., and the first time period is
between 1 and 30 minutes.
23. The method of claim 21 wherein the second temperature is
between -60.degree. C. and -200.degree. C., or is below -60.degree.
C.
24. The method of claim 21, wherein the first temperature is
between -2.degree. C. and the homogenous nucleation temperature of
water and the second temperature is below -60.degree. C.
25. The method of claim 24, wherein the second temperature is
between -60.degree. C. and -200.degree. C.
26. The method of claim 1 further comprising recovery of the
cryopreserved cells by thawing the cells to optimize cell
viability.
27. The use of cells cryopreserved using the method of claim 1 in
transplantation, diagnostics, or in vitro fertilization.
28. A method for optimizing a cryopreservation protocol for
cryopreserving cells wherein cells are cooled in such a way as to
maintain a constant amount of intracellular supercooling.
29. The method of claim 1 wherein the cells are stem cells.
30. The method of claim 29 wherein the amount of constant
supercooling is 10.degree. C.
31. The method of claim 29 wherein the amount of constant
supercooling is between 5.degree. C. and 20.degree. C.
32. The method of claim 28 wherein the amount of constant
supercooling is between 1.degree. C. and the homogenous nucleotide
temperature of water.
33. The method of claim 28 wherein the amount of constant
supercooling is between 1.degree. C. and 40.degree. C.
34. The method of claim 28 that does not comprise the use of
cryoprotectants.
35. The method of claim 28 that does not comprise the use of
permeating cryoprotectants.
Description
RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application No. 60/611,391 entitled "Method of
Cryopreserving Cells", having a current filing date of Sep. 18,
2004, but for which a petition was filed to correct the filing date
to Sep. 17, 2004; and to Canadian Patent Application Number
2,482,045, also entitled "Method of Cryopreserving Cells", filed
Sep. 17, 2004. All of such references are herein incorporated by
reference.
TECHNICAL FIELD
[0002] This application relates to methods of cryopreservation,
particularly methods of cryopreserving cells and tissues.
BACKGROUND
[0003] Cryobiology is the study of the effects of low temperatures
on biological systems. Although freezing is lethal to most living
systems, cryobiologists have been able to preserve cells and
tissues at a range of subzero temperatures, as low as liquid
nitrogen temperatures (-196.degree. C.). Currently, cryoprotection
can be applied to most cells in suspension, such as stem cells,
other progenitor cells, red and white blood cells, sperm cells,
oocytes, ova, and cellular materials derived from tissues and
organs (including but not limited to pancreatic islet cells,
chondrocytes, cells of neural origin, cells of hepatic origin,
cells of ophthalmolic origin, cells of orthopedic origin, cells
from connective tissues, cells of reproductive origin, and cells of
cardiac origin). Cryopreservation has also been used to effectively
preserve tissue, such as heart valves, embryos, skin, articular
cartilage, and islets of Langerhans and an increasing range of
engineered tissues and tissue constructs. Although the current
recovery of viable cells post-thaw may be sufficient for some
clinical uses, recovery is generally considered less than optimal
due to injury during the freezing process.
[0004] Cryopreservation has been applied to many cell and tissue
types. Recent developments in the utilization of a variety of stem
cells, including umbilical cord blood stem cells have revived
interest in optimizing cryopreservation techniques for cells and
tissues (D. Krause, 2002). In particular, for stem cells and other
cell types which are obtained in low numbers from donors, high
recovery of these cell types is crucial. High recovery is also
important in cryopreservation of engineered cells due to the high
cost and length of time for manufacturing such cells. Emerging
higher standards for cell and tissue banking (Guide to safety and
quality assurance for organs, tissues and cells, 2.sup.nd edition,
2004, Council of Europe Publishing, France), specifically stem cell
banking, will be required to meet future needs of cell banking and
therefore, optimal cryopreservation techniques are fundamental.
[0005] Currently, cryopreservation of cells has been most
successful with the use of cryoprotectants and cryopreservation of
stem cells has been most successful with the use of the permeating
cryoprotectant, dimethyl sulfoxide (DMSO). There are, however,
limitations to the use of DMSO. Adverse affects have been
associated with infusion of stem cells preserved with DMSO (Davis
et al., 1990; Egorin et al., 2001; Santos et al., 2003; Zambelli et
al., 1998). Some researchers have attempted to reduce the amount of
DMSO (Abrahamsen et al., 2002; Beaujean et al., 1998) or combine it
with a non-permeating cryoprotectant, such as Hydroxyethyl starch
(HES) (Donaldson, 1996; Halle et al., 2001; Katayama et al.,
1997).
[0006] In non-clinical studies to examine the effects of low
temperatures on cells, some cells have been cryopreserved without
the use of a specific cryoprotectant such as DMSO (Farrant et al,
1974; Knight, Farrant et al. 1977). However, cooling profiles were
not optimized and cell recoveries were not as high as with
cryoprotectants. These studies were used in research to understand
the mechanisms of cryoinjury and cryoprotection.
[0007] In current cryopreservation procedures, cells are generally
cooled at a constant rate which is optimized for the cell type and
cryoprotectant. This protocol has typically been approached
empirically by varying cooling rates and the nature and
concentration of cryoprotectants. In addition to cooling at a
constant rate, other techniques have been described to examine the
effects of low temperatures on cells, including a two-step freezing
technique. The two-step freezing technique (J. Farrant et al.,
1974) is a method to examine the effects of osmotic interactions on
cell recovery over a broad range of subzero temperatures. In this
procedure, lymphocytes were cooled rapidly to various subzero
temperatures and held for various periods of time before being 1)
thawed directly from that holding temperature or 2) rapidly cooled
to -196.degree. C. before thawing. McGann and Farrant later
reported that the subzero temperature and the length of hold time
at that temperature were factors to consider when attempting to
maximize cell survival (McGann and Farrant, 1976). To date an easy
method that can optimize cooling profiles for a cell type or for
various cell types for cryopreservation of cells is not available
and a reliable method that does not use cryoprotectants, especially
permeating cryoprotectants, has not been recommended, especially
for clinical use of the cells.
[0008] There is significant interest in designing an optimized
cryopreservation protocol for all cell types and tissues, which
maintains cell and tissue viability but does not require toxic
cryoprotectants. Further there is a need for protocols to
cyropreserve larger volumes of cells and tissues. Further there is
a need to develop a model or optimization protocol to optimize
cooling profiles to cryopreserve cells.
SUMMARY OF INVENTION
[0009] This invention relates to any non-linear cooling
cryopreservation method for cryopreserving cells and/or tissue that
is comprised of determining an optimal cooling profile for maximum
recovery of specific cells and tissues, and applying the cooling
profile to the respective cells and tissues. By non-linear cooling
cryopreservation method we mean throughout any cryopreservation
protocol for which, by design, temperature versus time is other
than a single straight line or a profile made of two line segments
with different slopes. In one embodiment, a non-linear cooling
cryopreservation method is achieved by a non-constant cooling rate
during at least a portion of the method. In another embodiment the
non-linear cryopreservation method is achieved by a two-step
cooling process, wherein the cells or tissue are cooled at a
constant or non-constant rate to a first holding temperature
(referred to throughout as hold temperature) and then subsequently
at a constant or non-constant rate to a second temperature
(referred to throughout as storage temperature).
[0010] The invention also relates to methods of optimizing
cryopreservation methods by determining an optimal non-linear
cooling profile and applying the profile to cryopreservation
methods. The invention also relates to cryopreservation methods
optimized by the method of the invention. This invention also
relates to any product prepared for transplantation or other uses
using such a protocol.
[0011] The optimal cooling profile is based in part or in whole on
profiles determined using a computer simulation of cellular or
tissue response during cooling and/or warming. The cellular
responses are predicted from mathematical models using cellular
osmotic transport properties, phase diagrams, and compostition and
thermodynamic parameters for the intra- and extra-cellular
solutions for a particular cell type.
[0012] In one embodiment applicable to any cell type or tissue
profiles are determined by requiring the amount of intracellular
supercooling to remain below a maximum value throughout the method
determined by correlating simulation predictions with viability
outcomes. Supercooling is the amount the temperature is below the
thermodynamic equilibrium freezing point of the solution. For
instance, in one embodiment, the maximum intracellular supercooling
is 10.degree. C., i.e the cells or tissues are cooled without
intracellular freezing or intracellular ice nucleation to a
temperature below the freezing point of the intracellular solution
but by not more than 10.degree. C.
[0013] In one embodiment of the invention, the method of
cryopreserving stem cells comprises cooling the cells or tissue at
a constant or non-constant rate to a first temperature (hold
temperature), holding at that temperature for a first period of
time (the hold time), then cooling the cells or tissue at a
constant or non-constant rate to a second temperature (storage
temperature) for storing the cells or tissue. In one embodiment the
hold time is between 1 to 30 minutes. In another embodiment the
hold time is between 1 to 10 minutes. In another embodiment it is
between 1 to 5 minutes. In yet another embodiment the hold time is
between 1 to 3 minutes.
[0014] The cryopreservation methods of the invention can be used
with cells stored without cryoprotectants. The protocols can also
be used with cells stored with cryoprotectants, including
permeating and non-permeating cryoprotectants. Such permeating
cryoprotectants include but are not necessarily limited to DMSO,
glycerol, propylene glycol and ethylene glycol. Such non-permeating
cryoprotectants include but are not limited to sugars, starches,
protein, serum, plasma or other macromolecules.
[0015] The invention can be applied to any types of cells,
including but not limited to stem cells, other progenitor cells,
red and white blood cells, sperm cells, oocytes, ova, cells for
research or transplant purposes, and cellular materials derived
from tissues and organs (including but not limited to pancreatic
islet cells, chondrocytes, cells of neural origin, cells of hepatic
origin, cells of ophthalmolic origin, cells of orthopedic origin,
cells from connective tissues, cells of reproductive origin, and
cells of cardiac origin). Throughout this application, cells
include cells organized as tissues. Stem cells include but are not
limited to human peripheral blood stem cells, human umbilical cord
blood stem cells and stem cells derived from tissues and solid
organs or other sources, including but not limited to fetal and/or
embryonic sources. The invention is not limited to human cell types
and is extendable to all mammalian and non-mammalian species.
[0016] In one embodiment, the method of the invention can be used
to cryopreserve stem cells, including but not limited to
hematopoietic stem cells, umbilical cord blood stem cells,
mesenchymal stem cells, stem cells derived from organs or tissues,
and stem cells grown in culture (eg. TF-1 cells). As such, in one
embodiment, the invention provides a stepped method of
cryopreserving stem cells that comprises cooling the cells to a
first temperature (hold temperature), holding at that first
temperature for a first period of time, then cooling the cells to a
second temperature for storing the cells.
[0017] In one embodiment of the invention, the amount of
supercooling and concentration of intracellular KCl were used as
predictors of cryoinjury.
[0018] Another embodiment of the invention includes any
unit/equipment used to execute the non-linear cryopreservation
protocols on cells in small or large samples or tissues. Including
but not limited to a bulk freezing unit or a cryomicroscopy
apparatus.
[0019] In one embodiment, the stem cells are cooled to a
temperature between -3.degree. C. and -30.degree. C. held for 1 to
30 minutes, then cooled to a temperature below -60.degree. C. to
store the cells. In one embodiment, the first temperature is
between -5.degree. C. and -15.degree. C. The hold time is between 1
to 3 minutes and the second temperature is below -60 .degree. C.
including but not limited to liquid Nitrogen temperatures.
[0020] In another embodiment, the invention provides a method of
restoring cryopreserved cells comprising warming the cells
cryopreserved using a method of the invention noted herein using
known protocols in the art, including but not limited to warming
rates greater than 25.degree. C./min, warming in a temperature
regulated bath at a preset temperature (0-40.degree. C.), and
warming at a constant rate greater than 25.degree. C./min. In one
embodiment the recovery of cells preserved by the methods of the
present invention without cryoprotectants are comparable with that
obtained using cryoprotectants. In one embodiment the recovery rate
is 75% with non-permeating cryoprotectants.
[0021] In one embodiment of the invention, the cryopreservation
methods of the invention and the cells and/or tissues recovered
from cryopreservation using the methods of the invention can be
used for research, transplantation, diagnostics and genetic
testing, cell/tissue banking for surveillance, toxicity testing and
for in vitro fertilization.
[0022] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples, while indicating preferred embodiments of the
invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
reading the detailed description.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1. Schematic of CryoSim5 program used to simulate
cellular responses.
[0024] FIG. 2. A representative cooling profile for TF-1 cells in
the stepped method, cooled from room temperature to -15.degree. C.
measured with a thermocouple (measured). The curve is fitted
according to Newton's Law of Cooling (fitted).
[0025] FIG. 3. Simulations of temperatures as a function of time
for TF-1 cells in the stepped method, cooled to various subzero
hold temperatures (-3.degree. C. to -40.degree. C.) using Newton's
Law of Cooling (constant=6.6 s.sup.-1) and held for 3 minutes,
prior to rapid cooling at 325.degree. C./min.
[0026] FIG. 4. Example simulations of volume as a function of
temperature for TF-1 cells in the stepped method, cooled according
to Newton's Law of Cooling (constant=6.6 s.sup.-1) to various
subzero hold temperatures (-3.degree. C. to -40.degree. C.) and
held at that temperature for (a) 0.5 min., (b) 3 min., and (c) 10
min., prior to rapid cooling at 325.degree. C./min.
[0027] FIG. 5. Examples of simulations of intracellular
supercooling as a function of temperature for TF-1 cells in the
stepped method, cooled according to Newton's Law of Cooling
(constant=6.6 s.sup.-1) to various subzero hold temperatures
(-3.degree. C. to 40.degree. C.) and held at that temperature for
(a) 0.5 min., (b) 3 min., and (c) 10 min., prior to rapid cooling
at 325.degree. C./min. Arrows indicate maximum supercooling.
[0028] FIG. 6. Example simulations of [KCl].sub.i as a function of
temperature for TF-1 cells in the stepped method, cooled according
to Newton's Law of Cooling (constant=6.6 s.sup.-1) to various
subzero hold temperatures (-3.degree. C. to -40.degree. C.) and
held at that temperature for (a) 0.5 min., (b) 3 min., and (c) 10
min., prior to rapid cooling at 325.degree. C./min. Arrows indicate
maximum [KCl].sub.i.
[0029] FIG. 7. Maximum [KCl].sub.i and maximum intracellular
supercooling as a function of temperature from example simulations
for TF-1 cells in the stepped method, cooled to various subzero
hold temperatures and held at that temperature for (a) 0.5 min. (b)
3 min. and (c) 10 min. prior to rapid cooling at 325.degree.
C./min.
[0030] FIG. 8. Maximum [KCl].sub.i and maximum supercooling as a
function of temperature from example simulations for TF-1 cells in
the stepped method, cooled to various subzero hold temperatures and
held at that temperature for 3 min. prior to rapid cooling at
325.degree. C./min. The shaded box indicates the optimal hold
temperature of between -4.degree. C. and -6.degree. C. based on
10.degree. C. maximum intracellular supercooling and 3 M maximum
intracellular [KCl].sub.i.
[0031] FIG. 9. Membrane integrity for TF-1 cells (.+-.SEM) in
serum-free RPMI media cooled using the stepped method to various
subzero hold temperatures from room temperature by immersion in a
constant-temperature bath, held 3 minutes, and then either thawed
directly (upper curve) or cooled rapidly at 325.degree. C./min in
liquid nitrogen (lower curve) before being thawed.
[0032] FIG. 10. The membrane integrity of TF-1 cells (.+-.SEM) in
serum-free RPMI media as a function of hold time for cells cooled
using the stepped method to (a) -5.degree. C. and (b) -25.degree.
C., held for a period of time before being either thawed directly
(upper curves) or cooled rapidly at 325.degree. C./min in liquid
nitrogen (lower curves) before being thawed.
[0033] FIG. 11. Contours of membrane integrity of TF-1 cells in
serum-free RPMI media after being cooled using the stepped method
to various subzero hold temperatures and held for a duration
ranging from 0.5 to 10 minutes before either (a) thawed directly or
(b) cooled rapidly at 325.degree. C./min in liquid nitrogen prior
to thawing.
[0034] FIG. 12. Membrane integrity of TF-1 cells in 10% DMSO cooled
at 1.degree. C./min to various subzero temperatures and then either
thawed directly (upper curve) or cooled rapidly at 325.degree.
C./min in liquid nitrogen (lower curve) before being thawed.
[0035] FIG. 13 Optimal non-linear cooling profiles calculated for
TF-1 cells using specified maximum amount of supercooling. The
calculated profile is that which would maintain the intracellular
supercooling at or below the specified amount as TF-1 cells are
cooled. In the embodiment shown here a maximum cooling rate has
been imposed until the cells first reach the specified amount of
intracellular supercooling.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0036] "Supercooling" is the amount the temperature is below the
thermodynamic equilibrium freezing point of the solution. For
instance, in one embodiment, the maximum intracellular supercooling
is 10.degree. C., i.e the cells or tissues are cooled without
intracellular freezing or intracellular ice nucleation, under
conditions where the difference between the temperature of the
sample and the freezing point of the intracellular solution never
exceeds 10.degree. C.
[0037] "Cooling profile" or "temperature profile" is the
specification of temperature as a function of time during the
cryopreservation process. The mathematical derivative of this
profile yields cooling rates at all time points during the protocol
and therefore specification of cooling rates and duration of
cooling can be determined from the cooling profile, conversely,
cooling rates and duration of cooling can be specified to obtain a
cooling profile over a particular time and temperature.
[0038] "Optimal cooling profile" is the cooling profile for maximum
recovery of the cells and applying the cooling profile to the
cells. The optimal cooling profile is determined in part or in
whole using a simulation of cellular responses to cooling
parameters. The cooling parameters comprise cell temperature,
duration of temperature exposure, temperature as a function of
time, and nature and concentration of cryoprotectants. The cellular
responses are determined from mathematical models of osmotic
transport properties, phase diagrams, and composition and
thermodynamic parameters for the intra- and extra-cellular
solutions for a particular cell type.
[0039] "Permeating Cryoprotectants" are cryoprotectants that can
penetrate the cell membrane and be present intracellularly.
Examples of permeating cryoprotectants include DMSO, glycerol,
propylene glycol and ethylene glycol. These cryoprotectants tend to
work by depressing the freezing point which lowers the temperature
at which ice is formed, and by reducing the amount of ice formed
which, in turn, lowers the temperature at which a specific
concentration of electrolytes occurs.
[0040] "Non-Permeating Cryoprotectants" are cryoprotectants that do
not penetrate the cell membrane and remain in the extracellular
solution. Examples of non-permeating cryoprotectants include high
molecular weight additives, such as sugars, starches, protein,
serum, plasma and other macromolecules. These cryoprotectants tend
to work by promoting osmotic water loss from cells at higher
subzero temperatures but contribute to osmotic stresses on the
cell.
[0041] "Cells" as used herein also includes cells organized into
tissues.
[0042] "Non-linear Cooling". In the context of the cryopreservation
method of the invention, Non-linear Cooling is meant that
throughout any cryopreservation protocol for which, by design,
temperature versus time is other than a single straight line or a
profile made of two line segments with different slopes. In one
embodiment, a non-linear cooling cryopreservation method is
achieved by a non-constant cooling rate during at least a portion
of the method. In another embodiment the non-linear
cryopreservation method is achieved by a two-step cooling process,
wherein the cells or tissue are cooled at a constant or
non-constant rate to a first holding temperature (referred to
throughout as hold temperature) and then subsequently at a constant
or non-constant rate to a second temperature (referred to
throughout as storage temperature).
[0043] "Two-Step Cooling" is a non-linear cooling method, wherein
the sample is cooled to a first hold temperature and maintained at
that temperature for a hold time before the sample is cooled to a
second storage temperature.
[0044] "Hold Time" is the time at which the sample is maintained at
the first hold temperature in the two-step cooling method.
[0045] "Hold Temperature" or "first temperature" in the two-step
cooling method is the temperature at which or the temperature range
within which the sample is maintained for the duration of the hold
time.
[0046] "Storage Temperature" is the temperature at which the cells
are stored. It can also be referred to as the "second temperature"
in the two-step-cooling process. In one embodiment the storage
temperature is below -60.degree. C. In another, the sample is
stored in liquid nitrogen. or anywhere in between these values.
Description
[0047] Throughout the following description, specific details are
set forth in order to provide a more thorough understanding of the
invention. However, the invention may be practiced without these
particulars. In other instances, well known elements have not been
shown or described in detail to avoid unnecessarily obscuring the
invention. Accordingly, the specification and drawings are to be
regarded in an illustrative, rather than a restrictive, sense.
[0048] This invention relates to any non-linear cooling
cryopreservation protocol for cryopreserving cells comprising of
determining an optimal cooling profile for maximum recovery of the
cells and applying the cooling profile to the cells. The optimal
cooling profile is determined in part or in whole using a
simulation of cellular responses to cooling parameters. The cooling
parameters comprise cell temperature, duration of temperature
exposure, temperature profile, and nature and concentration of
cryoprotectants. The cellular responses are determined from
mathematical models of osmotic transport, phase diagrams, and
compostition and thermodynamic parameters for the intra- and
extra-cellular solutions for a particular cell type.
[0049] The cryopreservation protocol can be used with cells stored
without cryoprotectants. The protocol can also be used with cells
stored with cryoprotectants, including permeating and
non-permeating cryoprotectants. Such permeating cryoprotectants
include but are not limited to DMSO, glycerol, propylene glycol and
ethylene glycol. Such non-permeating cryoprotectants include but
are not limited to sugars, starches, serum, protein, serum, plasma
or other macromolecules.
[0050] The invention can be applied to any types of cells,
including but not limited to stem cells, other progenitor cells,
red and white blood cells, sperm cells, oocytes, ova, cells for
research or transplant purposes, and cellular materials derived
from tissues and organs (including but not limited to pancreatic
islet cells, chondrocytes, cells of neural origin, cells of hepatic
origin, cells of ophthalmolic origin, cells of orthopedic origin,
cells from connective tissues, cells of reproductive origin, and
cells of cardiac origin). Throughout this application, cells
include cells organized as tissues. Stem cells include but are not
limited to human peripheral blood stem cells, human umbilical cord
blood stem cells and stem cells derived from tissues and solid
organs or other sources, including but not limited to fetal and/or
embryonic sources. The invention is not limited to human cell types
and is extendable to all mammalian and non-mammalian species. In
one embodiment of the invention, the non-linear cooling
cryopreservation protocol comprises cooling the cells to a first
temperature for a first period of time, then cooling the cells to a
storage temperature for a second period of time prior to
thawing.
[0051] In another embodiment of the invention, the non-linear
cooling cryopreservation protocol can be executed on cells using a
bulk freezing unit or a cryomicroscopy apparatus or other suitable
apparatus, including one with a programmable thermocycler, that can
be programmed to cool cells according to a particular cooling
profile, for instance to maintain intracellular supercooling below
a maximum amount or at a constant amount, or a profile that applies
the optimized two-step cooling protocol.
[0052] The invention also relates to methods of optimizing
cryopreservation protocols by determining an optimal non-linear
cooling profile and applying the profile to cryopreservation
protocols. The invention also relates to cryopreservation protocols
optimized by the method of the invention.
[0053] The invention also relates to non-linear cooling
cryopreservation protocols for cryopreserving stem cells.
[0054] In a specific embodiment of the invention, the method of
cryopreserving stem cells comprises cooling the stem cells to a
first temperature for a first period of time, then cooling the
cells to a second temperature for storing the stem cells. In one
embodiment, the stem cells are cooled to a temperature between
-3.degree. C. and -30.degree. C. held at that temperature for 1 to
30 minutes, then cooled to a temperature below -60.degree. C. to
store the cells.
Description of Simulation Tool
[0055] The present invention provides a method and simulation tool
that can be used to obtain cooling profiles and protocols for
cryopreserving cells. The method can be used to obtain cooling
profiles for cryopreserving cells that does not necessitate the use
of cryoprotectants. Instead of using cryoprotectants to alter the
properties of solutions so that cells may be cooled at a constant
rate (normally 1.degree. C./min) and achieve high recovery, in one
aspect of the invention, the inventors' approach (Ross-Rodriguez,
2003(a); Ross-Rodriguez et al., 2003(b)) has been to use the
properties of the intracellular and extracellular solutions and
osmotic transport parameters of subject cells to design optimal
cryopreservation protocols. A novel aspect of this approach is that
the temperature profile is not constrained to be linear. Rather,
the intracellular and extracellular solution properties, along with
cellular osmotic properties, are used to generate a temperature
profile that minimizes cryoinjury.
[0056] During the course of the inventors' research on cryoinjury
and cryoprotection, the inventors have developed a mathematical
model of cellular osmotic responses at low temperatures using real
(nondilute) solution assumptions for both the carrier solution and
the cellular cytoplasm (McGann and Elliott, 2003). In one
embodiment of the invention, this model has been implemented in a
computer program as a simulation tool to calculate intracellular
and extracellular parameters as the temperature changes and ice
forms or melts. The simulation tool can be used to generate an
optimized temperature profile for cooling the cells in order to
obtain maximal recovery of the cells upon thawing. The tool can be
applied to any cell type by using appropriate parameters of the
cells and solutions.
[0057] In some embodiments of the invention, simulations are based
on changes in the composition of the extracellular solution as
water is converted to ice during cooling, and the osmotic responses
of cells to these changes.
[0058] Features of the simulation include the use of the phase
diagram information for the extracellular and intracellular
solutions, the osmotic characteristics of the plasma membrane and
the temperature dependencies of the cellular osmotic parameters to
calculate the cellular osmotic responses to the concentration of
solutes in the residual liquid in the presence of ice at low
temperatures.
[0059] A computer program was developed (using the Delphi
programming language) to perform the simulations based on either
calculated or measured temperature profiles. The program also
includes a function to generate the temperature profile required to
maintain the cytoplasm at a constant degree of supercooling. One
embodiment of the present invention, is to experimentally determine
the maximum tolerable amount of intracellular supercooling, to use
this amount of supercooling in simulations to generate non-linear
temperature profiles for cryopreservation. In addition to the
resulting temperature profile, all calculated parameters are
reported as a function of time to allow access to both
intracellular and extracellular concentrations and fluxes.
Predictors of Cryoinjury
[0060] One embodiment of this invention uses the amount of maximum
intracellular supercooling as a predictor of cryoinjury related to
intracellular ice formation, and the maximum intracellular KCl
concentration as a predictor for cryoinjury related to exposure to
concentrated solutions.
Application of Method to Different Cell Types and Tissues
[0061] The invention can be applied to any types of cells,
including but not limited to stem cells, other progenitor cells,
red and white blood cells, sperm cells, oocytes, ova, cells for
research or transplant purposes, and cellular materials derived
from tissues and organs (including but not limited to pancreatic
islet cells, chondrocytes, cells of neural origin, cells of hepatic
origin, cells of ophthalmolic origin, cells of orthopedic origin,
cells from connective tissues, and cells of reproductive origin,
and cells of cardiac and cardiovascular origin). Stem cells include
human peripheral blood stem cells, human umbilical cord blood stem
cells, stem cells derived from tissues and solid organs or other
sources, including fetal and/or embryonic sources, as well as
mixtures of stem cells with other cells and from different sources.
Tissues include comea, cartilage, bone, skin, heart valves, Islets
of Langerhans, embryos from humans, animals, fish, shellfish and
plants, and ovarian tissues from humans and animals. The invention
is not limited to human cell types and is extendable to all
mammalian and non-mammalian species.
[0062] Applications of this invention to different cell types
utilize information on various cell parameters, including the
osmotic transport parameters and their temperature dependencies,
solution properties of the cytoplasm, solution properties of the
extracellular solution and any cryoprotectant or other solutes
present and the relationship between intracellular supercooling and
intracellular freezing of different cell types. Some of these
properties will not change significantly between different cells.
Solution properties, for example, depend primarily on the
concentrations of electrolytes and proteins, which are similar for
various types of cells. Similarly, the incidence of intracellular
freezing as a function of supercooling is likely to be similar for
different types of cells since there are likely similar mechanisms
of ice nucleation in cells, whether ice is initiated by spontaneous
nucleation in the cells, through aqueous pores in the plasma
membrane (Acker et al., 2001), by surface-catalyzed nucleation
(Toner, 1993), or by osmotic rupture of the plasma membrane (Muldew
and McGann, 1941). Conversely, the osmotic transport parameters and
their activation energies depend strongly on cell type and stage of
differentiation (McGrath, 1988).
Obtaining Cellular Osmotic Properties
[0063] Cellular osmotic properties can be obtained from the
literature (see for example Gao et al., 1998 and Hunt et al.,
2003). For cell types whose osmotic properties have not yet been
published, the osmotic properties can be measured as described by
the inventors in the examples and in the literature
(Ross-Rodriguez, 2003(a)).
Measuring Solution Properties
[0064] Calculations of osmotic transport at low temperatures
require descriptions of the intracellular and extracellular
solutions. Phase diagrams of binary and other solutions show that
solution behavior over the range of temperature between freezing
and -60.degree. C. is nonlinear, so an assumption of dilute
solutions is inappropriate. The constants in any equations that
describe non-dilute behavior are therefore required for the major
intracellular and extracellular solutes. This information has been
gathered for particular cell types from the literature.
Intracellular and extracellular solute information for cell types
can be gathered empirically, from the literature, and from the
inventors' own previous work. (MacMillan and Mayer 1945, Freedman
and Hoffman 1979, Elliott et al., 2002).
Monitoring Cryoinjury
[0065] Several in vitro methods can be applied to assess stem cell
recovery after experimental treatment and to further optimize hold
temperatures and hold times. These assessments may include but are
not limited to membrane integrity, metabolic and other functional
assays and/or colony growth in culture, and fluorescent assays,
such as SYTO/EB as noted in the examples below.
Infusible Extracellular Compounds
[0066] The inventors demonstrate that temperature profiles can be
generated to avoid intracellular freezing and to reduce cryoinjury
related to exposure to high concentrations of solutes, similar to
results for human lymphocytes (Farrant et al., 1974). However, to
simultaneously meet these criteria for some cells, i.e. conditions
required to avoid intracellular freezing may already subject the
cells to lethal exposure to the solution it may be optimal to use
infusible extracellular compounds. In this case further embodiments
of the invention include use of infusible extracellular compounds,
including but not limited to sugars, starches, such as Pentastarch
and hydroxyl starch, serum, proteins or plasma as
cryoprotectants.
Cryopreservation Protocol for Stem Cells
[0067] A two-step freezing protocol used by Farrant et al. to
obtain high recovery of human lymphocytes cryopreserved in serum
alone (Farrant et al., 1974) was optimized using simulation of a
stem cell line (TF-1 cells, a hematopoietic stem cell line) without
cryoprotectant (Ross-Rodriguez, 2003) and further optimized, and
validated using experimental measurements of post-thaw cell
recovery. Osmotic transport parameters were measured for the TF-1
cells and used in simulations of the two-step cooling protocol. The
maximum degree of intracellular supercooling over the course of the
cooling protocol was used as a predictor of cryoinjury due to
intracellular freezing, and the maximum intracellular potassium
chloride concentration used as a predictor of cryoinjury due to
exposure to the concentrated solutes. Minimum values for the
predictors of cryoinjury from the simulation indicated the range of
hold temperatures and hold times where cell recovery was expected
to be maximal in the absence of cryoprotectants.
[0068] Experimental measurement of TF-1 cell recovery using
membrane integrity showed maximal recovery in the hold temperature
range predicted by the simulations and low recovery at hold
temperatures outside the predicted range. The maximum recovery of
TF-1 cells without cryoprotectant thawed from -196.degree. C. was
equivalent to the recovery after conventional cryopreservation
(cooling at 1.degree. C./min in the presence of 10% DMSO). In a
specific embodiment, the zone of hold temperatures (-3.degree. C.
to -30.degree. C.), when held for 1-30 minutes, confer comparable
protection against injury to the standard 10% DMSO solution using
conventional cooling profiles.
[0069] Results of these experiments supported the concept of using
theoretical predictors of cryoinjury in the use of simulations to
reduce empirical experimentation in optimization of
cryopreservation protocols. These results also demonstrate the
value of simulations to be used in protocol design.
EXAMPLES
[0070] The following examples are intended to illustrate various
embodiments of the invention and are intended to be interpreted in
a non-limiting sense.
[0071] This invention relates to the cryopreservation of cells.
More particularly it relates to non-linear cooling methods for the
cryopreservation of cells. In another embodiment, the invention
relates to non-linear cooling methods for the cryopreservation of
cells that do not require the use of cryoprotectants for cell
recovery optimization, more particular permeating cryoprotectants
such as DMSO, glycerol, propylene glycol and ethylene glycol that
can have toxic, damaging osmotic, harmful or other undesired
effects on recovered cells especially cells for clinical use.
[0072] In yet another embodiment the invention relates to the
optimization of non linear cooling cryopreservation of cells by
taking into account: [0073] (a) the effects of intracellular
freezing on cell viability; and [0074] (b) solution effects on cell
viability; and [0075] (c) dependence of the above factors on time
and temperature [0076] (d) osmotic parameters for the extracellular
solution and for the intracellular solution for a particular cell
type.
[0077] In one embodiment, the invention provides a method to
optimize cooling profiles for cryoprotecting cells comprising:
[0078] (a) determining the osmotic parameters of the cell type,
including osmotically-inactive fraction, hydraulic conductivity and
its Arrhenius activation energy; [0079] (b) determining the
thermodynamic phase diagram of the intracellular and extracellular
solutions [0080] (c) determining the amount of intracellular
supercooling that leads to intracellular freezing (e.g. maximum
supercooling); [0081] (d) using the osmotic parameters of the cell
type (a), the thermodynamic phase diagram of the intracellular and
extracellular solutions (b), and maximum supercooling amounts (c)
under various conditions to model the cellular response to low
temperatures based on preferred selected criteria, eg. setting the
maximum amount of (i) intracellular supercooling to a desired
level, for instance does not exceed 10.degree. C. (for 2 step or
constant supercooling profiles) and/or (ii) intracellular
concentration of KCl to a desired level, for instance does not
exceed 3M; [0082] (e) optionally empirically testing the
simulations experimentally to compare with theoretical data; and
[0083] (f) optionally repeating steps (a) to (e) to adjust for
instance constants and values of osmotic and thermodynamic
properties of the cells and to further optimize the cooling
profile.
[0084] In order to make a simulation of a cooling cryopreservation
profile, in one embodiment, internal and external solution
compositions, as well as the values of cellular osmotic properties
(e.g. L.sub.p, E.sub.A of L.sub.p, V.sub.iso, V.sub.b,
.pi..sub.iso, .pi..sub.i, .pi..sub.e) are used. These properties
can be obtained from the literature (see for example Gao et al.,
1998 and Hunt et al., 2003) or by experimentation. For cell types
whose osmotic properties have not yet been published, the osmotic
properties can be measured as described by the inventors in the
examples and in the literature (Ross-Rodriguez, 2003(a)). As well,
the thermodynamic solution properties (K1, B2.sub.i, B3) can also
be obtained from the literature, from experimentation or from
existing experimental data (Bannerman, Elliott et al. 2005).
[0085] In one embodiment, the invention relates to a method of
optimizing cooling profiles for cells by obtaining, e.g. through
experimentation or prior literature, the values of one or more or
all of the following: [0086] (a) L.sub.p=hydraulic conductivity
[0087] (b) E.sub.A=activation energy of L.sub.p [0088] (c)
V.sub.iso=isotonic volume [0089] (d) V.sub.b=osmotically inactive
fraction [0090] (e) .pi..sub.isoisotonic osmolality [0091] (f)
.pi..sub.i=intracellular osmolality [0092] (g)
.pi..sub.e=extracellular osmolality; [0093] (h) B2.sub.i,
B3.sub.i=the second and third, respectively, osmotic virial
coefficients for each solute, i, and inserting the values into
thermodynamic equations, such as the following coupled equations
and obtaining a mathematical solution that can be used for cooling
profile simulations for various solution parameters or cell
types.
[0094] A range of equations can be used in particular simulations.
A person skilled in the art would appreciate that a variety of
equations may be used to obtain the desired osmotic and
thermodynamic properties noted above and represented in the
equations below. This invention is intended to encompass any and
all such equivalents.
[0095] In one embodiment, the equations include one or more of the
following: [0096] 1. The Jacobs and Steward model (Jacobs and
Steward 1932) dV/dt=L.sub.pART(.pi..sub.i-.pi..sub.e) [0097] where
V is the water volume in the cell (.mu.m.sup.3), t is the time
(min), L.sub.p is the hydraulic conductivity or rate at which water
moves across the cell membrane, A is the cell surface area
(.mu.m.sup.2), R is the universal gas constant (kcal/mol/K), T is
the absolute temperature (K), .pi..sub.e is the extracellular
osmolality (osmoles/kg water) and .pi..sub.i is the intracellular
osmolality (osmoles/kg water). L.sub.p can be determined using
measurements of the kinetics of volume change over time when
exposed to anisotonic solutions. This equation provides a function
of a change in volume over time in light of cell membrane
characteristics (e.g. hydraulic conductivity) for a particular cell
type and osmality of the internal and external solutions. A
suitable replacement membrane transport model could also be used
instead of this equation. [0098] 2. The Boyle van't Hoff
relationship (Lucke and McCutcheon 1932) V eq V iso = .pi. o .pi.
.times. ( 1 - v b ) + v b ##EQU1## [0099] where V.sub.eq is the
equilibrium volume (.mu.m.sup.3) of the cell, V.sub.iso is the
isotonic volume (.mu.m.sup.3), .pi..sub.o is the isotonic
osmolality (osmoles), .pi. is the experimental osmolality
(osmoles), and .nu..sub.b is the osmotically-inactive fraction.
Through graphical analysis of V.sub.eq/V.sub.iso as a function of
.pi..sub.o/.pi., .nu..sub.b can be determined by extrapolating the
line by linear regression to the y-intercept. This equation is used
to calculate the osmotically-inactive fraction, that fraction of
the cell volume that is not involved in the osmotic activities of
the cell, by expressing the equilibrium cell volume after exposure
to anisotonic solutions of impermeant solutes. A person skilled in
the art would appreciate that a replacement equilibrium equation
could also be used. [0100] 3. The Arrhenius temperature dependence
of Lp (Voet and Voet 1995) L p = L P o exp .function. ( - E a R T )
##EQU2## where L.sub.p.sup.o is a fitting constant, R is the
universal gas constant (kcal/mol/K) and T is the absolute
temperature (K). E.sub.A is the Arrhenius activation energy of
L.sub.p and is used to describe the temperature dependence of
L.sub.p. A person skilled in the art would appreciate that
replacement temperature dependence for the hydraulic conductivity
could also be used. [0101] 4. A specified Temperature as a function
of time, i.e. T=F(t) such as prescribed cooling rates at different
times or with a function or functions including but not limited to
either an experimentally determined numerical function or for
certain portions an analytical equation such as Newton's Law of
Cooling: (Incopera and Dewitt 2002) d T d t = k .DELTA. .times.
.times. T ##EQU3## [0102] where dT/dt is the rate of change in
temperature change in the sample with time, k (s.sup.-1) is the
fitting constant obtained from experimental data (Ross-Rodriguez
2003), and .DELTA.T is the difference in temperature between the
bath and the sample. A person skilled in the art would appreciate
that a replacement cooling profile could also be used.
Alternatively the cooling profile can be left as an adjustable
function obtained as an output of a simulation. [0103] 5a. The
Bannerman-Elmoazzen-Elliott-McGann equation (Elliott et al., 2002;
Bannerman et al.,submitted, 2005). .pi. = i = 1 .times. .times.
.times. .times. n .times. .times. [ m i + B2 i m i 2 + B3 i m i 3 +
j = i + 1 .times. .times. .times. .times. n .times. .times. [ ( B2
i + B2 j ) m i m j ] ] ##EQU4## [0104] where .pi. is osmolality of
the solution, m is the molality of the solute, and B2 and B3 are
fitting constants for specific solutes in water. For electrolytes,
the molality is multiplied by an empirically determined
"dissociation" constant. The constant B3 is non-zero only for
solutes with highly nonlinear behaviour, such as macromolecules. A
replacement description of the solution phase diagrams, ie.
freezing point as a function of solution composition and osmolality
as a function of composition, could also be used. [0105] 5b. The
freezing point of the intracellular or extracellular solution,
T.sub.FP, can be described by the equation (Elliott et al., 2002))
T.sub.FP=K.pi. [0106] where K is the molal freezing point
depression constant for water, and .pi. is the osmolality of the
solution, [0107] 6. The amount of intracellular supercooling (S) at
each instant is then given by S=T-T.sub.FP.
[0108] With temperature as a function of time (such as equation 4)
as an input, Equations 1,2,3, 5, 6 form a coupled set of
differential equations that can be solved, for instance, by a
computer yielding cell volume, molality of each impermeant
intracellular component, freezing point of the intracellular
solution and amount of supercooling each as a function of time
during the cooling profile. Since the cooling profile is specified,
there is a one-to-one correspondence of temperature and time so
that one also has then cell volume, molality of each impermeant
intracellular component, freezing point of the intracellular
solution and amount of supercooling each as a function of time
during the cooling profile.
[0109] In an alternative embodiment, the cooling profile can be
left unspecified, for instance by omitting equation 4. and
specifying the amount of supercooling, S, in equation 6. The
coupled equations 1,2,3,5,6 can then be solved yielding temperature
as a function of time as an output along with cell volume, molality
of each intracellular component, freezing point of the
intracellular solution, each as a function of time during the
cooling profile. Applying such a temperature profile to the cells
would keep the cells below a maximum amount of intracellular
supercooling as the cooling proceeds. Example profile for TF-1
cells calculated to restrict intracellular supercooling to
specified values are shown in FIG. 13. In one embodiment, a maximum
practical cooling rate has been imposed until the cells first reach
the specified amount of intracellular cooling.
[0110] In one embodiment, a person skilled in the art, to obtain a
solution to the set of Equations would appreciate that in addition
to known values of the parameters, (a)-(h), molar volumes of all
components, a mass or mole balance and unit conversions as
appropriate would be used to obtain the cooling profile. In one
embodiment, the set of equations can be solved on a commercially
available (or programmed by someone skilled in the art)
differential and algebraic equation solving computer program.
[0111] The method of the invention can also be used to develop a
cooling profile when there is a cryoprotectant, such as a
permeating cryoprotectant. A similar set of equations can be used
when there is a permeating solute (such as a permeating
cryoprotectant like DMSO). In this case, the set of equations
consists of 1-6 with an additional equation describing the permeant
solute membrane mass transport.
[0112] For instance, the membrane mass transport equation for
permeable solutes may be, for example [Jacobs, 1933; Jacobs et.
al., 1932] d N s i d t = P s .times. A .function. ( m s e - m s i )
##EQU5## where N.sub.s.sup.i is the number of moles of
intracellular solute, Ps is the membrane permeability to that
solute, m.sub.s.sup.e and m.sub.s.sup.i are respectively the
extracellular and intracellular molality of the solute or a
replacement equation, including but not limited to extensions of
equation 7 to account for non-idealities of solutions (Elmoazzen,
et. al., 2004); and/or Kedem-Katachlsky type reflection coefficient
(Kedem et.al, 1958).
[0113] Again, either [0114] i) temperature profiles, such as
described in equation 4, are given as an input and then 1,2,3,5,6
plus the additional permeant solute membrane mass transport
equation, equation 7, are solved to determine the amount of
supercooling as a function of time, (as well as cell volume,
molality of each intracellular component, and freezing point of the
intracellular solution and intracellular supercooling each as a
function of time); and/or [0115] ii) the temperature profile is
omitted and the amount of supercooling in equation 6 is set to a
constant, maximum value, and then equation 1,2,3,5,6 plus the
additional permeant solute membrane mass transport equation,
equation 7, are solved yielding the optimal temperature as a
function of time to be followed as the optimal cooling profile (as
well as cell volume, molality of each intracellular component, and
freezing point of the intracellular solution each as a function of
time).
[0116] The result from this method is a cooling profile
corresponding to prescribed conditions, or a set of cooling
profiles corresponding to a set of prescribed conditions.
[0117] In one embodiment, experiments, following the specified
temperature profiles are performed, the viability of the cells
after the application of the protocol is assessed and the
experimental results can be used
[0118] To determine: [0119] (i) what amount of supercooling if
reached at anytime during the a cooling protocol is tolerable by a
particular cell type without intracellular ice damage; and/or
[0120] (ii) what amount of constant supercooling yields a
temperature profile that is tolerable by the cells; and/or [0121]
(iii) what concentration of intracellular solutes (such as KCl) if
reached at anytime during the cooling profile (maximum KCl) is
tolerable by the cells, and to further optimize the cooling profile
and simulation by adjusting fitting constants and the like.
[0122] As an example of the one embodiment of the invention, for
instance, for TF-1 cells in the sample calculations shown, the
isotonic solution composition (a) and osmotic parameters (b), the
cooling profiles and the solution thermodynamic parameters for TF-1
cells as listed in Table 1 can be inputted into the simulation
protocol and coupled equations to determine an optimal cooling
profile for a cell type, for instance at a preferred maximum
supercooling amount and KCl concentration throughout the profile.
The cooling profile would include rate of cooling over range of
time, including any hold times and hold temperatures taking into
account change of volume over time, change of temperature over time
and other changes in osmotic or thermodynamic parameters over
time.
[0123] In one embodiment, the method of the invention can be used
to determine optimal cryopreservation solution parameters and to
develop cryopreservation solutions. Such solutions can be optimized
for particular cell types and tissues.
[0124] In one embodiment the invention provides a method for
establishing cooling profiles of supercooling versus time and [KCl]
versus time and selecting those protocols which meet a desired
requirement on intracellular supercooling and intracellular [KCl]
for example those parameters that provide for cooling profiles
wherein the supercooling amount does not exceed 10 .degree. Celsius
and intracellular KCl concentration does not exceed 3M or obvious
chemical equivalents of these amounts, e.g. +/-10%. However a
person skilled in the art can use the method and vary the maximum
amounts of supercooling and KCl concentration depending on desired
cell viability or cell recovery. In one embodiment the invention
can provide cell recovery of 75% or more with the use of
non-permeating cryoprotectants after thawing. In another
embodiment, the invention can provide cell recovery without
cryoprotectants that are comparable to those obtained with
cryoprotectants, such as permeating and/or non-permeating
cryoprotectants. The method of the invention also involves methods
of non-linear cryopreserving cells and recovering cryopreserved
cells. Known methods can be used to recover cells cryopreserved
using the methods of the present invention, such as warming rates
greater than 25.degree. C./min, warming in a temperature regulated
bath at a preset temperature (0-40.degree. C.), and warming at a
constant rate greater than 25.degree. C.
[0125] In the method of the invention experimental parameters can
be inserted into equations to obtain constants for various cell
types and solution/condition parameters. Equations then can be used
to develop simulated, or in one embodiment optimal, cooling
profiles for different solutions for that particular cell type. In
one embodiment, cooling profiles are selected that limit the
maximum amount of supercooling (as an indicator of intracellular
freezing), and KCl concentration (as an indicator of solute effects
and cell viability) to optimal levels. In another embodiment, cell
volume over a particular cooling profile can be used to select for
desired cooling profiles, one that permits some equilibrium with
extraceullar solution conditions and dehydration to limit the
amount of water within the cells and minimize intracellular
freezing, while preventing too much dehydration that would
adversely effect cell viability.
[0126] In one embodiment, the amount of supercooling does not
exceed 10.degree. C. or obvious chemical equivalents thereof. In
another embodiment, the optimal concentration of intracellular KCl
does not exceed 3M or obvious chemical equivalents thereof. In one
embodiment, the invention relates to any cooling profile which is
based on restricting the amount of intracellular supercooling
during cooling to below a specified amount, e.g. 10.degree. C.;
and/or the intracellular solute concentration to below a specified
amount, e.g. 3M KCl. However, the maximum amount of intracellular
supercooling tolerable by a certain cell type in a certain
circumstance may vary, for example, between 1.degree. C. and
40.degree. C. and the maximum amount of intracellular solute
concentration represented by [KCl]i, tolerable by a certain cell
type in a certain circumstance may vary, for example, between 1 M
and 8M.
[0127] In one embodiment, the cells are cooled to a temperature
between -3.degree. C. and -30.degree. C. held for 1 to 30 minutes,
then cooled to a temperature below -60.degree. C. to store the
cells. In another embodiment, the cells are cooled to a temperature
of between -5.degree. C. and -15.degree. C., for a hold time
between 1 to 5, or in another embodiment 1 to 3 minutes and then
cooled to a second or storage temperature below -60.degree. C.
including but not limited to liquid Nitrogen temperatures. Again,
in one embodiment, it should be noted that the cells can be held at
a constant temperature within the desired range throughout the
desired hold time or at a non constant temperature within the
range. In another embodiment, cells are cooled to the hold
temperature in 0.1 to 5 minutes using linear or nonlinear profiles.
In yet another embodiment, cells are cooled from the hold
temperature to the second or storage temperature in 0.1 to 5
minutes using linear or nonlinear profiles
[0128] The above variables can be adjusted with different solutions
and the presence and absence of cryoprotectants, such as
permeating, non-permeating or a mixture of permeating and
non-permeating cryoprotectants. Examples of permeating
cryoprotectants include but are not necessarily limited to DMSO,
glycerol, propylene glycol and ethylene glycol. Examples of
non-permeating cryoprotectants include but are not necessarily
limited to sugars, starches, proteins, serum, plasma and other
macromolecules.
[0129] In another embodiment, a two-step cooling profile is used,
wherein cells are cooled to a first temperature (at either a
constant or non-constant rate i.e. following a linear or non-linear
temperature profile) and held for a desired period of time to
enable equilibration with extracellular solutions, while minimizing
intracellular freezing and solution effects and then cooling to a
second temperature or storage temperature at a constant or
non-constant rate (or following a linear or non-linear temperature
profile), that enables high recovery of cells recovered from the
storage temperature.
[0130] A person skilled in the art would appreciate that similar
methods can be used to determine optimal cooling cryopreservation
profiles for tissues.
Example 1
Theoretical Design of a Cryopreservation Protocol
1.1 Introduction
[0131] The cellular responses to the formation of ice in
surrounding solution are largely dependent on the movement of water
across the plasma membrane. Ice formation causes osmotic imbalance
across the cell membrane forcing water out of the cell to maintain
equilibrium with the extracellular solution. The properties of the
cell membrane, specifically the osmotic parameters, as well as the
solution thermodynamics of the intracellular and extracellular
solutions govern these changes in cell volume. The osmotic
parameters can be used in simulations to theoretically model
cellular responses to low temperatures. Simulations also provide
precise results regarding changes in cell volume and the amount of
supercooling. These results can then be used for comparisons
between cryopreservation protocols and for comparison between
different cell types which may be present in one tissue.
Ultimately, simulations allow for unlimited theoretical protocols
to be explored by controlling cooling and warming conditions, hold
times, hold temperatures, and the components of the intracellular
and extracellular compartments for any cell type for which the
osmotic parameters are known (Ross-Rodriguez, 2003).
[0132] To distinguish between the two types of injury, solution
effects and intracellular ice formation, the inventors simulated
the empirical procedure of two-step freezing.
[0133] The objective of this example was to determine the
theoretical responses of TF-1 cells, a model for hematopoietic stem
cells (HSC) (Kitamura, 1989(a); Marone, 2002), to subzero hold
temperatures and to hold times at those temperatures. Simulations
were done using the osmotic parameters of TF-1 cells reported in
Ross-Rodriguez, 2003. The objective of these simulations was to
theoretically determine the conditions of TF-1 cells at various
stages of a freezing protocol. Maximum levels of intracellular
electrolyte concentrations ([KCl].sub.i) and of supercooling were
examined upon cooling the cells with the two-step protocol as
predictors for solution effects injury and intracellular ice
formation injury, respectively.
1.2 Simulations of Two-Step Freezing Protocol
Methods
[0134] Simulations were performed according to those done in
Ross-Rodriguez, 2003 using the osmotic parameters of TF-1 cells
(Table 1) in the CryoSim5 program (Dr. Locksley McGann, University
of Alberta, Canada). The simulations were based on a two-step
freezing technique, which has been used to examine the effects of
high solute concentrations and intracellular ice formation on cell
survival during freezing (Farrant, 1977). The cryopreservation
protocol was defined by assigning a starting temperature and then
varying the cooling rates, based on typical two-step freezing
procedures. Supercooling and [KCl].sub.i were used as predictors of
potential intracellular ice formation and solution effects,
respectively. Coupled equations 1-6, noted above were used to
obtain the simulations and the results noted below.
Temperature Profiles
[0135] The two-step freezing technique involved rapidly cooling the
samples to various subzero hold temperatures before either being
thawed directly in a 37.degree. C. water bath or cooled rapidly in
liquid nitrogen first and then thawed (McGann, 1979). For the
simulations, cells were cooled using a temperature profile derived
from Newton's Law of Cooling. Newton's Law of Cooling describes the
temperature change in response to heat transfer which depends on
the difference in temperature between the bath and the sample.
Alternatively, more sophisticated heat transfer modeling could be
used if needed (Incropera & Devitt, 2002). A fitting constant
in Newton's Law of Cooling was determined by monitoring the cooling
profile of a sample taken from room temperature and exposed to the
experimental subzero temperature with a Type T thermocouple (Omega,
Laval, Canada). FIG. 2 is a representative cooling profile of a
sample cooled from room temperature to -15.degree. C. This profile
was then fitted to a curve and the equation was then used in
simulations. The variations between the experimental and fitted
curves were due to the latent heat of fusion. Alternatively a
numerical representation of the experimental curve could be
used.
[0136] Simulations were performed in which cells with no
cryoprotectant were cooled to various subzero hold temperatures
ranging from -3.degree. C. to the homogenous nucleation temperature
of water, or in one embodiment to -40.degree. C. and held at that
temperature for 0.3, 0.5, 0.7, 1, 2, 3, 5, 7, or 10 minutes, prior
to being cooled at 325.degree. C./min to the storage temperature
(Ebertz, 2002).
1.3 Results
Changes in Cell Volume During Cooling
[0137] FIG. 3 shows simulation results of temperature as a function
of time for TF-1 cells cooled to various subzero hold temperatures
ranging from -3.degree. C. to -30.degree. C., held for various hold
times (minutes), prior to being cooled at 325.degree. C./min to the
storage temperature Based on the results from the simulations, the
hold times were grouped according to similarities of changes in
cell volume, [KCl].sub.i, and supercooling: Hold times of 1 minute
or less will be represented by the 0.5 minute results; hold times
between 2 and 5 minutes will be represented by the 3 minute
results; and hold times of between 7 and 10 minutes will be
represented by the 10 minute results. FIG. 4 demonstrates the
changes in cell volume as a function of temperature upon cooling to
various subzero hold temperatures ranging from -3.degree. C. to
-30.degree. C., holding for a duration, and then cooling at
325.degree. C./min to the storage temperature. The results shown
are for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times. Cells
showed a progressive decrease in cell volume upon cooling. Cells
only held for 0.5 minutes at the subzero temperature did not reach
the same volumes as those held for 3 or 10 minutes at -3.degree. C.
and -35.degree. C. These results suggest that the cells have not
had sufficient amount of time to dehydrate with a hold time of 0.5
minutes, as opposed to hold times greater than 3 minutes, for both
high and low subzero hold temperatures. This data also indicates
that the cells would have a higher amount of supercooling at these
outlying hold temperatures due to the lack of cellular dehydration.
Also, with lower concentrations of [KCl].sub.i, it is possible that
the cells would not be subjected to high solution effects.
Supercooling During Cooling
[0138] FIG. 5 demonstrates the changes in amount of intracellular
supercooling as a function of temperature upon cooling to various
subzero hold temperatures ranging from -3.degree. C. to -30.degree.
C., prior to being cooled rapidly at 325.degree. C./min. Results
shown are for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times.
Supercooling of up to 10.degree. C. occurs for all the hold times
at hold temperatures down to -12.degree. C. This indicates that
supercooling plays a key role in potential injury during freezing
to lower subzero hold temperatures. At these lower temperatures,
cells were exposed to increasingly supercooled conditions of up to
30.degree. C. of supercooling at -40.degree. C.
[KCl].sub.i During Cooling
[0139] FIG. 6 demonstrates the changes in [KCl].sub.i as a function
of temperature upon cooling to various subzero hold temperatures
ranging from -3.degree. C. to -30.degree. C., prior to being cooled
rapidly at 325.degree./min. The results shown are for (a) 0.5 min.,
(b) 3 min., and (c) 10 min. hold times. Cells cooled to lower
subzero hold temperatures showed increasing concentrations of
[KCl].sub.i, with the highest concentration for cells cooled to
-30.degree. C. and held for greater than 3 minutes. This correlates
with the gradual decrease in cell volume reported in the previous
section. This gradual increase in [KCl] demonstrates the potential
for increased solution effects upon cooling to the lower subzero
hold temperatures. The data show similar concentrations of
[KCl].sub.i at all hold temperatures except at -3.degree. C. and
below -30.degree. C.
Maximum supercooling and [KCl].sub.i During Cooling
[0140] The maximum amount of supercooling was calculated as the
highest amount of supercooling which occurred throughout the
cooling profile for each hold temperature. FIG. 5a shows calculated
amount of supercooling as a function of temperature for TF-1 cells
with arrows indicating where the maximum supercooling was
determined for the various hold temperatures. The maximum amount of
supercooling was calculated and graphed as a function of hold
temperature (FIG. 7). Results are shown for (a) 0.5 min., (b) 3
min., and (c) 10 min. hold times. The maximum supercooling obtained
appears to be the primary contributor to potential injury, which
suggests that a target hold temperature between -6.degree. C. to
-12.degree. C. would lead to high levels of survival because the
supercooling does not exceed 10.degree. C. Cells held for 0.5
minutes have a more narrow range of optimal hold temperatures,
limited by the amount of supercooling. These results correlate with
the lack of cellular dehydration discussed in the previous
sections.
[0141] The maximum amount of [KCl].sub.i was calculated as the
highest concentration of intracellular KCl which occurred
throughout the cooling profile for each hold temperature. FIG. 6a
shows the [KCl].sub.i as a function of temperature for TF-1 cells,
with arrows indicating where the maximum [KCl].sub.i was determined
for the various hold temperatures. The levels of maximum
[KCl].sub.i for cells held for 0.5, 3 and 10 minutes gradually
increase from -3.degree. C. to -20.degree. C. (FIG. 7). The slope
between -3.degree. C. and -6.degree. C. varies for cells held for
0.5 minutes and 3 to 10 minutes, suggesting that at hold
temperatures between -3.degree. C. and -6.degree. C., there may be
a difference in cell recovery between hold times of 0.5 minutes and
3 to 10 minutes. For all the hold times, based on the temperature
range set by the 10.degree. C. limit to supercooling, the results
suggest that the lower [KCl].sub.i levels would result in better
cell recovery. FIG. 8 shows the hold temperature ranges for cells
held for 3 minutes based on 10.degree. C. supercooling and 3 M
[KCl].sub.i. This range varies between the 0.5 minute hold time and
the 3 and 10 minute hold time. Based on simulations a target hold
temperature of approximately -6.degree. C. should result in the
highest cell recovery for all the hold times.
[0142] These simulations suggest that supercooling plays a key role
in two-step freezing and the effects of increasing solute
concentrations are secondary. The optimal hold temperature for the
cells is a function of the amount of time spent to cool to a
specific temperature, which influences [KCl].sub.i and
intracellular supercooling.
Example 2
Experimental Correlation and Optimization of a
Theoretically-Designed Cryopreservation Protocol
2.1 Introduction
[0143] The simulations performed in Example 1 predicted that
subzero hold temperature and time spent at that temperature were
critical variables in the optimization of cryopreservation
protocols. In order for simulations to be used in cryopreservation,
the predictions of simulations were tested empirically. The purpose
of this example was to explore the range of subzero hold
temperatures and time spent at those temperatures. Two-step cooling
experiments were conducted with TF-1 cells and cooling profiles
leading to high or low survival were compared with those that were
theoretically predicted in Example 1 to have high or low cell
survival. Membrane integrity was used as an assay for freeze-thaw
injury. Cooling profiles leading to high or low recovery were
compared with those that were theoretically predicted.
2.2 Materials & Methods
TF-1 Cell Culture
[0144] TF-1 cells (ATCC, Manassas, Va.) were grown at 37.degree. C.
in 5% CO.sub.2 in RPMI 1640 Medium Modified (ATCC) with 10% fetal
bovine serum (FBS) (ATCC), and supplemented with 2 ng/mL
recombinant human GM-CSF (Stemcell Technologies, Vancouver,
Canada). Cells were maintained between 0.1.times.10.sup.6 and
1.times.10.sup.6 cells/mL, according to ATCC guidelines. Prior to
experiments, cells were washed twice with serum-free RPMI media and
incubated overnight. Cells were then centrifuged and re-suspended
at a concentration of 4.times.10.sup.6 cells/mL, which was
necessary for the viability assessment program to be used.
Experimental Solutions
[0145] TF-1 cells were re-suspended in serum-free RPMI prior to the
two-step freezing experiments.
Two-Step Freezing Experiments
[0146] Samples of 0.2 mL cell suspension, in serum-free RPMI, in
glass tubes were allowed to equilibrate at room temperature for 5
minutes. Control samples were either warmed in a 37.degree. C.
water bath or cooled rapidly in liquid nitrogen. Experimental
samples were individually transferred into a methanol bath preset
at -3, -6, -9, -12, -15, -20, -30, and 40.degree. C. and allowed to
equilibrate for 2 minutes at that temperature prior to ice
nucleation with cold forceps. After nucleation, samples were
allowed to equilibrate for 3 minutes before either being thawed
directly in a 37.degree. C. water bath or cooled rapidly in liquid
nitrogen. Samples were kept in liquid nitrogen for a minimum of 1
hour prior to being thawed in a 37.degree. C. water bath. Duplicate
samples were used for both the direct thaw and the liquid nitrogen
conditions at each hold temperature. Each experiment was repeated
in triplicate.
[0147] The two-step freezing experiments were repeated with varying
hold times. Cells were cooled to a hold temperature of -5, -7, -9,
-12, -15, or -25.degree. C. and allowed to equilibrate for 2
minutes prior to ice nucleation with cold forceps. After
nucleation, samples were allowed to equilibrate for 0.5 to 10
minutes before either being thawed directly in a 37.degree. C.
water bath or cooled rapidly in liquid nitrogen. Samples were kept
in liquid nitrogen for a minimum of 1 hour prior to being thawed in
a 37.degree. C. water bath. Duplicate samples were used for both
the direct thaw and the liquid nitrogen conditions at each hold
temperature. Each experiment was repeated in triplicate.
Viability Assessment
[0148] Cell viability was assessed by a membrane integrity assay.
The assay was performed by incubating cells with SYTO.RTM. 13
(Molecular Probes, Eugene, Oregon) and ethidium bromide (EB)
(Sigma, Mississauga, Canada) (Yang, 1998). Syto 13 permeates the
cell membrane of all cells and complexes with DNA and it fluoresces
green under UV exposure. EB penetrates cells with a damaged plasma
membrane and also complexes with DNA fluorescing red under UV
conditions. The dual stain allows for differentiation between cells
with and without intact plasma membranes.
[0149] The Syto/EB stain was prepared using 40 .mu.L of 2.5 mM EB
stock solution and 10 .mu.L of 5 mM Syto.RTM.13 stock solution
mixed with 350 .mu.L 1.times. phosphate-buffered saline (PBS).
Final concentrations were 0.25 mM EB and 0.125 mM Syto. Twenty
.mu.L of stain was added to each sample and allowed to incubate for
2 minutes at room temperature. Fluorescent images were captured
using a Leitz Dialux 22 fluorescence (440-480 nm) microscope
(Leitz, Germany) fitted with a PIXERA DiRactor (Pixera Corporation,
Los Gatos, Calif., USA) digital camera. The Viability Assessment
Program (The Great Canadian Computer Company, Spruce Grove,
Canada), which counts red versus green pixels was used to quantify
cell membrane integrity from digital images (Jomha, 2003). This
method measures membrane integrity of the cell remaining after
experimental treatment.
2.3 Results
Varying Hold Temperature
[0150] TF-1 cells were suspended in serum-free RPMI, cooled to
various hold temperatures down to -40.degree. C. and held for 3
minutes, prior to being thawed directly or cooled rapidly in liquid
nitrogen (FIG. 9). Cells cooled in liquid nitrogen showed
comparable results for membrane integrity to cells directly thawed
from temperatures ranging from -15.degree. C. to -40.degree. C.
Cells thawed directly from the hold temperatures showed a 50%
decrease in membrane integrity by -17.degree. C., indicating that a
major portion of cells were damaged prior to being cooled rapidly
in liquid nitrogen (FIG. 9). However, this membrane damage occurred
at a higher subzero hold temperature for cells cooled at
0.9.degree. C./min. The maximum recovery of 58.8.+-.6.5% was seen
for TF-1 cells cooled to -12.degree. C. or -15.degree. C. before
being cooled rapidly in liquid nitrogen. This recovery was
comparable with 63.7% recovery obtained after conventional cooling
at a constant rate of 0.9.degree. C./min in 10% DMSO/RPMI.
Varying Experimental Hold Time
[0151] TF-1 cells were cooled to hold temperatures of -5, -7, -9,
-12, -15, or -25.degree. C. and allowed to equilibrate for 2
minutes prior to ice nucleation with cold forceps. After
nucleation, samples were held at that temperature for varying times
(0.3, 0.5, 0.7, 1, 2, 3, 5, 7, and 10 minutes) before either being
thawed directly in a 37.degree. C. water bath or cooled rapidly in
liquid nitrogen. FIG. 10a shows the membrane integrity of TF-1
cells as a function of hold time for cells cooled to -5.degree. C.,
held and cooled rapidly in liquid nitrogen. Comparable results of
membrane integrity were obtained for cells held at -7.degree. C. to
-9.degree. C. (results not shown). However there was minimal
membrane integrity for cells held below -25.degree. C. (data shown
in FIG. 10b). Cells that were directly thawed from the subzero hold
temperatures after being held showed progressive decrease in
membrane integrity based on reduced temperature and increased
duration of hold time (FIG. 11a). Results indicated a high
percentage of membrane integrity of 55 to 60%, when cells were held
for 1-5 minutes at high subzero hold temperatures. Cells cooled
from room temperature to -5.degree. C. and -7.degree. C. and held
for 1-3 minutes, prior to plunging into liquid nitrogen resulted in
the highest percentage of membrane integrity of approximately 60%
(FIG. 11b). A hold time of greater than 5 minutes resulted in a
marked decrease in cell survival. This data indicates that there is
a zone of subzero hold temperatures (-5.degree. C. to -15.degree.
C.), when held for 1-3 minutes, which confers protection against
injury comparable to DMSO.
[0152] With larger sample volumes, different cell types and
different compositions of the intracellular and extracellular
solutions, hold temperatures and hold times may vary over larger
ranges, for instance 1 to 30 minutes between -3 and -30.degree. C.
For instance, in the present examples 200 microlitre samples were
used. If one uses larger volumes, such 50 ml standard bags for
umbilical cord blood samples, the latent heat of fusion effects
will differ and may deviate from Newton's Law of Cooling, which
would effet hold times. In which case numerical values of the
temperature of the sample as a function of time would be obtained
for the temperature profile, by for instance putting a thermocouple
into the bag.
5.4 Correlation with Theoretically-Designed Protocol
Discussion of Experimental Results
[0153] The experimental results for cryopreserving TF-1 cells
without cryoprotectants indicate that cells can be cryopreserved
without DMSO. This data indicates that there is a zone of subzero
hold temperatures (-5.degree. C. to -15.degree. C.), when held for
1-3 minutes, which confers comparable protection against injury to
the standard 10% DMSO/RPMI solution, previously reported in
Ross-Rodriquez. This range would constitute an optimal subzero
temperature range for these hold times based on experimental
results.
Comparison of Theoretical and Experimental Results
[0154] Simulations were done based on an empirical approach to
cryopreservation, two-step freezing, which can be used to examine
the role of exposure to subzero hold temperatures and exposure
time. The cooling rates used in the two-step freezing protocol are,
in some instances, governed by Newton's Law of Cooling and were
determined experimentally in Example 1. Cells were exposed to
increasingly supercooled conditions up to 30.degree. C. of
supercooling at a temperature of -40.degree. C. Supercooling
appears to be a primary indicator of potential freezing injury due
to intracellular freezing. In these experiments, the inventors
found that beyond 10.degree. C. of supercooling, cell viability was
not optimized for TF-1 cells. The proposed target hold temperature
was suggested to be between -4.degree. C. and -12.degree. C., as
supercooling was restricted to less than 10.degree. C., which is
comparable to the range determined empirically. Also, based on
levels of intracellular KCl ([KCl].sub.i), it was suggested that
the higher subzero hold temperature would have the lowest potential
for solution effects based on the lack of cell dehydration, which
was also supported by this data.
[0155] Two-step freezing experiments demonstrated a high percentage
of membrane integrity for TF-1 cells when cells were cooled to
between -5.degree. C. and -12.degree. C. and held for 1-5 minutes.
These hold temperatures corresponded with the theoretical values of
5.degree. C. to 10.degree. C. supercooling, which suggested that a
certain amount of supercooling is necessary to achieve a higher
viability (Diller, 1975). However, this also supports the belief
that excessive supercooling may lead to damage as a result of
intracellular ice formation.
[0156] Based on the simulations, the duration of time the cells
were held at the subzero hold temperature was also considered an
important factor. When cells were held for 0.5 minutes, they did
not have sufficient time to dehydrate and reach the same volume as
cells held for greater than 2 minutes. This excess intracellular
water may have caused damage by forming ice upon subsequent
cooling. According to the two-step freezing experiments, cells held
for 2 minutes at -5.degree. C. and for 5 minutes at -12.degree. C.,
had the highest cell recovery. Those held for 10 minutes may have
been exposed to high concentrations of solutes for a duration which
was damaging. Simulations from Example 1 predicted that there was
no difference in [KCl].sub.i concentrations and supercooling
between hold times down to -25.degree. C. The experimental results
demonstrated that the differences in membrane integrity between the
hold times may depend on the duration of exposure, which is
consistent with the theoretical results.
[0157] For all the hold times, simulations predicted a progressive
increase in [KCl].sub.i upon cooling to lower hold temperatures
down to -25.degree. C. for cells held for 0.5 minutes. The
experimental results for cells thawed directly from subzero hold
temperatures demonstrated a decline in membrane integrity with
decreasing hold temperature. At low subzero hold temperatures
(<-20.degree. C.), cells directly thawed had low percentages of
membrane integrity (<30%). Therefore, either the exposure time
and/or the concentration of solutes may have been significant
variables for freezing injury.
[0158] As will be apparent to those skilled in the art in the light
of the foregoing disclosure, many alterations and modifications are
possible in the practice of this invention without departing from
the spirit or scope thereof.
[0159] While the present invention has been described with
reference to what is presently considered to be a preferred
embodiment, it is to be understood that the invention is not
limited to the disclosed embodiment. To the contrary, the invention
is intended to cover various modifications and equivalent
arrangements included within the spirit and scope of the appended
claims. It should be noted the application is intended to encompass
obvious chemical equivalents of the cells, tissues or other
components or parameters of the invention as described herein,
which are equivalents that produce the same or equivalent desired
result for a particular feature of the invention.
[0160] All publications, patents, and patent applications are
herein incorporated by reference in their entireties, to the same
extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety. TABLE-US-00001 TABLE 1
a) Isotonic solution composition Extracellular Intracellular NaCl
0.171 molal 0.010 molal KCl 0.005 molal 0.128 molal Protein 0 0.004
molal Total Osmolality 0.301 osm/kg 0.301 osm/kg b) Osmotic
parameters Isotonic volume 776 .mu.m.sup.3 Inactive fraction 0.353
L.sub.p at 20.degree. C. 0.342 .mu.m/min/atm Activation Energy for
L.sub.p 13.4 kcal/mol c) Cooling profiles k 6.6 s.sup.-1 d)
Solution thermodynamic parameters K.sub.diss B2.sub.i
(molal.sup.-1) B3 (molal.sup.-2) NaCl 1.702 0.0299 -- KCl 1.742 --
-- Protein -- 49.3 3.07 .times. 10.sup.4
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