CN115039762B - Placental stem cell storage solution and storage method - Google Patents
Placental stem cell storage solution and storage method Download PDFInfo
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- CN115039762B CN115039762B CN202210695535.XA CN202210695535A CN115039762B CN 115039762 B CN115039762 B CN 115039762B CN 202210695535 A CN202210695535 A CN 202210695535A CN 115039762 B CN115039762 B CN 115039762B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
The invention provides a storage solution and a storage method for placenta stem cells, wherein the storage method for the placenta stem cells comprises the steps of dispersing the placenta stem cells in a HEPES buffer solution of trehalose, then crosslinking the dispersed placenta stem cells with a calcium chloride HEPES buffer solution containing sodium hyaluronate and galactose through a high-pressure electrospray device to form coated particles, reducing the load of a dry cell membrane in a freezing process due to the effects of sodium alginate, sodium hyaluronate and galactose, maintaining the balance of the dry cells, and then freezing the cells in a freezing solution of human albumin, 3% -10% of trehalose, 1% -3% of galactose and 2% -8% of sodium hyaluronate. The placenta stem cell storage method avoids the use of a cytotoxic refrigerant dimethyl sulfoxide, and improves the cell survival rate and the differentiation and regeneration capacity of the stem cells after the stem cells are frozen and stored compared with a vitrification freezing method.
Description
Technical Field
The invention relates to the field of stem cell freezing storage, in particular to a placental stem cell storage solution and a storage method.
Background
Long term storage of rare, unique or patient-specific cell types and their ready availability are of great importance in regenerative medicine. Among the methods known today for preserving vital cells, cryopreservation, by assessing cell viability, membrane integrity and function after thawing, is a method that has acceptable results and is universally evaluated. Key cryopreservation parameters include (i) cryoprotectant type and cryoprotectant concentration, (ii) equilibration temperature and time, (iii) cooling/thawing rate and (iv) temperature of intracellular/extracellular ice formation during freezing. Unless optimal parameters are applied, this may result in low viability and recovery of cells after cryopreservation and loss of plasticity and pluripotency. The standard freezing method for most of these stem cell lines uses a slow cooling rate at 10% (v/v) dimethyl sulfoxide (Me) 2 SO) as cryoprotectant and fetal bovine serum (10-20%, v/v) at various concentrations. In the reproductive medicine field, "ultra-rapid cooling" or vitrification has been successfully used to freeze germ cells and tissues. However, the use of vitrification for the storage of single stem cells in solution or loose cell complexes remains disadvantageous, mainly due to the cytotoxic effects of high concentrations of these refrigerants, such as dimethyl sulfoxide, which is detrimental to cell recovery.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a placental stem cell storage solution and a storage method.
In order to realize the purpose, the invention adopts the technical scheme that:
a method of placental stem cell storage, the method comprising the steps of:
(1) Dispersing the placenta stem cells in 8-15 mM HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) buffer solution and uniformly mixing with sodium alginate to obtain mixed solution A, wherein the concentration of the placenta stem cells in the mixed solution A is (0.5-2) multiplied by 10 6 cell/mL, the mass concentration of the sodium alginate in the mixed solution A is 1-2%;
(2) Injecting the mixed solution A into a solution B through a high-pressure electric spraying device, stirring and crosslinking for 12-18 minutes, wherein the solution B comprises 8-15 mM HEPES and 50-300 mM CaCl 2 0.5-1% by weight of sodium hyaluronate and 0.3-0.8% by weight of galactose;
(3) Using 8-15 mM HEPES and 1.0-2.0 mM CaCl 2 Washing the product in the step (2) by the formed buffer solution, and collecting particles with the particle size of 180-400 mu m;
(4) Suspending the particles obtained in the step (3) in a refrigerating fluid for freezing, wherein the refrigerating fluid comprises human serum albumin with the mass concentration of 0.5% -2%, trehalose with the mass concentration of 3% -10%, galactose with the mass concentration of 1% -3% and sodium hyaluronate with the mass concentration of 2% -8%.
The method for storing the placental stem cells disperses the placental stem cells in a HEPES buffer solution containing trehalose, and then the placental stem cells are crosslinked with a calcium chloride HEPES buffer solution containing sodium hyaluronate and galactose by a high-pressure electrospray device to form coated particles, so that the load of a stem cell membrane in a freezing process is reduced under the action of sodium alginate, sodium hyaluronate and galactose, the balance of the stem cells is maintained, and the stem cells are frozen in a freezing solution containing human albumin, 3% -10% of trehalose, 1% -3% of galactose and 2% -8% of sodium hyaluronate. The placenta stem cell storage method avoids the use of a cytotoxic refrigerant dimethyl sulfoxide, and improves the cell survival rate and differentiation and regeneration capacity of the stem cells after frozen storage compared with a vitrification freezing method.
Preferably, the parameters for stirring and crosslinking by injecting the mixed solution a into the solution B through a high-pressure electrospray device include: the distance between the positive electrode and the negative electrode is 8-12 cm, the flow rate of the mixed liquid A is 8-12 ml/h, and the working voltage is 18-22 kV.
Preferably, the method for stirring and crosslinking by injecting the mixed solution a into the solution B through a high-pressure electrospray device comprises the following steps: and (3) putting the mixed solution A into an injector, fixing the injector on an injection pump of the high-voltage electric spraying device, adjusting the distance between the positive electrode and the negative electrode of the high-voltage electric spraying device, setting the speed of the injection pump, adjusting the working voltage and starting the injection pump.
Preferably, in the step (4), the particles in the step (3) are suspended in the refrigerating fluid and are balanced at 4 ℃ for 30-50 minutes, and are cooled down to-78 to-82 ℃ at a cooling rate of 0.8-1.2 ℃/min for cold storage.
Preferably, in the step (1), the concentration of the placental stem cells in the mixture a is (0.8 to 1.2) × 10 6 cells/mL.
The research of the invention finds that the concentration of the placental stem cells in the mixed solution A is (0.8-1.2) multiplied by 10 6 When the cells are in a/mL state, the dispersibility of the cells is better, and the particle size distribution of the particles after the electrospray crosslinking coating is more uniform.
Preferably, the solution B comprises 8-15 mM HEPES and 80-150 mM CaCl 2 0.6-0.8% by weight of sodium hyaluronate and 0.3% by weight-0.8% by weight of galactose.
The invention researches find that when the component concentration of the solution B is in the range, the particle size distribution of the particles coated by electrospray crosslinking is more uniform, the particle size is concentrated on 220-330 mu m, and the cell survival rate and the differentiation and regeneration capacity are relatively higher after freezing and thawing.
Preferably, 8-15 mM HEPES and 1.0-2.0 mM CaCl are used in the step (3) 2 Washing the product in the step (2) by using the buffer solution, and collecting particles with the particle size of 220-330 mu m.
The invention also provides a placenta storage solution, which comprises a solution A, a solution B and a solution C;
the solution A is a HEPES buffer solution dissolved with sodium alginate, the concentration of HEPES in the HEPES buffer solution is 8-15 mM, and the mass concentration of sodium alginate in the buffer solution is 1% -2%;
the liquid bag BComprises 8 to 15mM HEPES and 50 to 300mM CaCl 2 0.5-1% by weight of sodium hyaluronate and 0.3-0.8% by weight of galactose;
the C liquid comprises human serum albumin with the mass concentration of 0.5-2%, trehalose with the mass concentration of 3-10%, galactose with the mass concentration of 1-3% and sodium hyaluronate with the mass concentration of 2-8%.
Preferably, the B solution comprises 8-15 mM HEPES, 80-150 mM CaCl 2 0.6-0.8% by weight of sodium hyaluronate and 0.3% by weight of galactose.
The placenta storage solution can be prepared into a reagent and is convenient to use.
The invention has the beneficial effects that:
the invention provides a storage solution and a storage method for placenta stem cells, wherein the storage method for the placenta stem cells comprises the steps of dispersing the placenta stem cells in a HEPES buffer solution of trehalose, then crosslinking the dispersed placenta stem cells with a calcium chloride HEPES buffer solution containing sodium hyaluronate and galactose through a high-pressure electrospray device to form coated particles, reducing the load of a dry cell membrane in a freezing process due to the effects of sodium alginate, sodium hyaluronate and galactose, maintaining the balance of the dry cells, and then freezing the cells in a freezing solution of human albumin, 3% -10% of trehalose, 1% -3% of galactose and 2% -8% of sodium hyaluronate. The placenta stem cell storage method avoids the use of a cytotoxic refrigerant dimethyl sulfoxide, and improves the cell survival rate and the differentiation and regeneration capacity of the stem cells after the stem cells are frozen and stored compared with a vitrification freezing method.
Drawings
Fig. 1 is a schematic view of a high-pressure electrospray device in the placental stem cell storage method according to an embodiment of the present invention.
The device comprises a high-voltage device 1, a high-voltage device 2, an injection pump 3, an injector 4, an injector pipeline 5, a positive electrode 6, a positive electrode and negative electrode distance 7, a negative electrode 8, a solution B container 9 and an electromagnetic stirring device.
FIG. 2 is a graph showing the comparison of the storage effect of the storage method of placental stem cells according to the embodiment of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
The placenta storage solution provided by the embodiment of the invention comprises a solution A, a solution B and a solution C;
the solution A is a HEPES buffer solution dissolved with sodium alginate, the concentration of HEPES in the HEPES buffer solution is 12mM, and the mass concentration of sodium alginate in the buffer solution is 1.5%;
the solution B comprises 12mM HEPES and 120mM CaCl 2 0.6% by weight sodium hyaluronate and 0.5% by weight galactose;
the C liquid comprises human serum albumin with the mass concentration of 1%, 5% trehalose, 1.5% galactose and 4% sodium hyaluronate.
The placental stem cell storage method provided by the embodiment of the invention comprises the following steps:
(1) Dispersing the placenta stem cells in 12mM HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) buffer solution, and mixing with sodium alginate to obtain mixture A with placenta stem cell concentration of 1 × 10 6 cell/mL, the mass concentration of the sodium alginate in the mixed solution A is 1.5%;
(2) Injecting the mixed solution A into a solution B through a high-pressure electric spraying device, stirring and crosslinking for 12-18 minutes, wherein the solution B comprises 12mM HEPES and 120mM CaCl 2 0.6% by weight sodium hyaluronate and 0.5% by weight galactose;
(3) Using 8-15 mM HEPES and 1.0-2.0 mM CaCl 2 Washing the product in the step (2) by using the buffer solution, and collecting particles; detecting a small amount of sample by a particle size distribution instrument, wherein the particle size distribution is between 250 and 310 mu m;
(4) Suspending the particles obtained in the step (3) in a refrigerating fluid for freezing, wherein the refrigerating fluid comprises human serum albumin with the mass concentration of 1%, 5% trehalose, 1.5% galactose and 4% sodium hyaluronate; suspending the particles obtained in the step (3) in a refrigerating fluid, balancing for 40 minutes at 4 ℃, and cooling to-80 ℃ at a cooling rate of 1 ℃/min for cold storage.
As shown in fig. 1, the parameters for stirring and crosslinking the mixed solution a into the solution B by the high-pressure electrospray apparatus include: the distance between the positive electrode and the negative electrode is 10cm, the flow rate of the mixed liquid A is 10ml/h, and the working voltage is 20kV.
The method for injecting the mixed solution A into the solution B through a high-pressure electric spraying device to stir and crosslink comprises the following steps: the mixed solution A is contained in an injector 3, the injector 3 is fixed on an injection pump 2 of a high-voltage electric spraying device, the distance (6) between a positive electrode and a negative electrode (5,7) of the high-voltage electric spraying device 1 is adjusted, the speed of the injection pump 2 is set, the solution B is placed in a solution B container, stirring is kept through an electromagnetic stirring device, the working voltage is adjusted, and the injection pump 2 is started.
Example 2
As an embodiment of the present invention, the only difference between this embodiment and embodiment 1 is: the solution B comprises 12mM HEPES and 50mM CaCl 2 0.6% by weight sodium hyaluronate and 0.5% by weight galactose.
Collecting particles; a small amount of samples are taken and detected by a particle size distribution instrument, and the particle size distribution is between 220 and 280 mu m.
Example 3
As an embodiment of the present invention, the only difference between the present embodiment and embodiment 1 is: the solution B comprises 12mM HEPES and 80mM CaCl 2 0.8 wt% of sodium hyaluronate and 0.5 wt% of galactose.
Collecting particles; a small amount of samples are taken and detected by a particle size distribution instrument, and the particle size distribution is 235-290 mu m.
Example 4
As an embodiment of the present invention, the only difference between this embodiment and embodiment 1 is: the solution B comprises 12mM HEPES and 150mM CaCl 2 0.6% by weight sodium hyaluronate and 0.5% by weight galactose.
Collecting particles; a small amount of samples are taken and detected by a particle size distribution instrument, and the particle size distribution is between 250 and 300 mu m.
Example 5
As a method for storing placental stem cells according to an embodiment of the present invention,the only difference between this example and example 1 is: the solution B comprises 12mM HEPES and 250mM CaCl 2 0.6 wt% sodium hyaluronate and 0.5 wt% galactose.
Collecting particles; a small amount of samples are taken and detected by a particle size distribution instrument, and the particle size distribution is between 190 and 380 mu m.
Example 6
As an embodiment of the present invention, the only difference between this embodiment and embodiment 1 is: the solution B comprises 12mM HEPES and 150mM CaCl 2 1% by weight of sodium hyaluronate and 0.5% by weight of galactose.
Collecting particles; a small amount of samples are taken and detected by a particle size distribution instrument, and the particle size distribution is between 200 and 380 mu m.
Comparative example 1
A placental stem cell storage method of a comparative example of the present invention, comprising the steps of:
(1) Dispersing the placental stem cells in 12mM HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) buffer solution and uniformly mixing with sodium alginate to obtain mixed solution A, wherein the concentration of the placental stem cells in the mixed solution A is 1 × 106 cells/mL, and the mass concentration of the sodium alginate in the mixed solution A is 1.5%;
(2) The mixed solution A is injected into a solution B and stirred for 12 to 18 minutes, wherein the solution B comprises 12mM HEPES and 120mM CaCl 2 0.6% by weight sodium hyaluronate and 0.5% by weight galactose;
(3) Freezing the mixed solution in the step (2) in a freezing solution, wherein the freezing solution comprises human serum albumin with the mass concentration of 1%, trehalose with the mass concentration of 5%, galactose with the mass concentration of 1.5% and sodium hyaluronate with the mass concentration of 4%; and (4) suspending the particles obtained in the step (3) in a refrigerating fluid, balancing for 40 minutes at 4 ℃, and cooling to-80 ℃ at a cooling rate of 1 ℃/min for cold storage.
Comparative example 2
A placental stem cell storage method of a comparative example of the present invention, comprising the steps of: dispersing the placenta stem cells into refrigerating fluid, wherein the refrigerating fluid comprises human serum albumin with the mass concentration of 1%, 5% trehalose, 1.5% galactose, 4% sodium hyaluronate, 5% dimethyl sulfoxide and 12mM HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) buffer solution; dispersing the placenta stem cells in refrigerating fluid, balancing at 4 deg.C for 40 min, cooling to-80 deg.C at a cooling rate of 1 deg.C/min, and storing.
Examples of the experiments
1. After programmed temperature thawing of multifunctional stromal cells from the amniotic membrane of common macaque placenta, the cells were cultured in Dulbecco's modified Eagle's sterile medium (DMEM) containing 15% (v/v) fetal bovine serum, 1% penicillin/streptomycin, and 1% ascorbic acid in a tissue culture dish at 37 ℃ and in a 5% carbon dioxide humidified incubator.
2. At passage 9, digestion was performed using 0.05%/0.02% (w/v) trypsin/EDTA solution, centrifugation at 20g for 5 min, and removal of the liquid. Resuspended in 12mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid) buffer solution for further use.
3. (1) the above-treated ninth generation cells were stored for 15 days using the placental stem cell storage methods of examples 1-6 and comparative examples 1-2, respectively.
(2) And (3) heating the stored cell sample to-20 ℃ at the heating rate of 2 ℃/min, putting the cell sample into an ice-water mixture at the temperature of 0 ℃ for thawing for 20 minutes, and thawing for 45 minutes in a constant-temperature incubator at the temperature of 37 ℃.
(3) The sample treated in the above step (2) was immersed in a 55mM sodium citrate solution, followed by gentle shaking for 5 minutes. Cells were harvested by centrifugation at 80g for 5 minutes and resuspended in fresh medium.
By 0.4% trypan blue staining method and Vi-
XR Cell Viability Analyzer (Cell Analyzer counter) measures Cell membrane integrity.
The ninth generation of cells treated as above was used as a blank control by 0.4% trypan blue staining and Vi-
XR Cell Viability Analyzer (Cell Analyzer counter) measures Cell membrane integrity.
Cell membrane integrity (%) = (total cell count-stained cell count)/total cell count × 100%.
As shown in fig. 2, it can be seen from fig. 2 that the placental stem cell storage method according to the present invention can significantly protect the integrity of cell membranes during the freezing process, and the survival rate of cells during the cryopreservation process is higher.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for placental stem cell storage, comprising the steps of:
(1) Dispersing the placenta stem cells in 8-15 mM HEPES buffer solution and uniformly mixing with sodium alginate to obtain mixed solution A, wherein the concentration of the placenta stem cells in the mixed solution A is (0.5-2) multiplied by 10 6 cell/mL, the mass concentration of the sodium alginate in the mixed solution A is 1-2%;
(2) Injecting the mixed solution A into a solution B through a high-pressure electric spraying device, stirring and crosslinking for 12-18 minutes, wherein the solution B comprises 8-15 mM HEPES and 50-300 mM CaCl 2 0.5-1% by weight of sodium hyaluronate and 0.3-0.8% by weight of galactose;
(3) Using 8-15 mM HEPES and 1.0-2.0 mM CaCl 2 Washing the product obtained in the step (2) by using the buffer solution, and collecting particles with the particle size of 180-400 mu m;
(4) Suspending the particles obtained in the step (3) in a refrigerating fluid for freezing, wherein the refrigerating fluid comprises human serum albumin with the mass concentration of 0.5% -2%, trehalose with the mass concentration of 3% -10%, galactose with the mass concentration of 1% -3% and sodium hyaluronate with the mass concentration of 2% -8%;
in the step (4), the particles in the step (3) are suspended in refrigerating fluid and balanced at 4 ℃ for 30-50 minutes, and the temperature is reduced to minus 78-minus 82 ℃ at the cooling rate of 0.8-1.2 ℃/min for cold storage.
2. The placental stem cell storage method according to claim 1, wherein the parameters for agitation crosslinking by high pressure electrospray device by infusion of mixed solution a into solution B comprise: the distance between the positive electrode and the negative electrode is 8-12 cm, the flow rate of the mixed liquid A is 8-12 ml/h, and the working voltage is 18-22 kV.
3. The placental stem cell storage method according to claim 1 or 2, wherein the method for cross-linking by stirring mixed solution A into solution B by high-pressure electrospray device comprises: and (3) putting the mixed solution A into an injector, fixing the injector on an injection pump of the high-voltage electric spraying device, adjusting the distance between the positive electrode and the negative electrode of the high-voltage electric spraying device, setting the speed of the injection pump, adjusting the working voltage and starting the injection pump.
4. The method for storing placental stem cells according to claim 1, wherein in step (1), the concentration of placental stem cells in mixture A is (0.8 to 1.2). Times.10 6 cells/mL.
5. The placental stem cell storage method according to claim 1, wherein solution B comprises 8 to 15mM HEPES, 80 to 150mM CaCl 2 0.6-0.8% by weight of sodium hyaluronate and 0.3% by weight-0.8% by weight of galactose.
6. The placental stem cell storage method according to claim 5, wherein 8 to 15mM HEPES and 1.0 to 2.0mM CaCl are used in step (3) 2 Washing the product in the step (2) by using the buffer solution, and collecting particles with the particle size of 220-330 mu m.
7. A placental storage solution, wherein said placental storage solution comprises solution A, solution B, solution C;
the solution A is a HEPES buffer solution dissolved with sodium alginate, the concentration of HEPES in the HEPES buffer solution is 8-15 mM, and the mass concentration of sodium alginate in the buffer solution is 1% -2%;
the solution B comprises 8-15 mM HEPES and 50-300 mM CaCl 2 0.5% by weight-1% by weight of sodium hyaluronate and 0.3% by weight0.8% wt galactose;
the C liquid comprises human serum albumin with the mass concentration of 0.5-2%, trehalose with the mass concentration of 3-10%, galactose with the mass concentration of 1-3% and sodium hyaluronate with the mass concentration of 2-8%.
8. The placental storage solution of claim 7, wherein solution B comprises 8-15 mM HEPES, 80-150 mM CaCl 2 0.6-0.8% by weight of sodium hyaluronate and 0.3% by weight-0.8% by weight of galactose.
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