CN107581186A - Cells frozen storing liquid and preparation method and application - Google Patents
Cells frozen storing liquid and preparation method and application Download PDFInfo
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- CN107581186A CN107581186A CN201710959578.3A CN201710959578A CN107581186A CN 107581186 A CN107581186 A CN 107581186A CN 201710959578 A CN201710959578 A CN 201710959578A CN 107581186 A CN107581186 A CN 107581186A
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Abstract
The present invention provides a kind of cells frozen storing liquid for being used safely in clinic and preparation method and application.The cells frozen storing liquid is made up of human serum albumins, dimethyl sulfoxide (DMSO), high polymer material and culture medium, foreign protei or serum are free of in cells frozen storing liquid, greatly reduce the security of clinical cytology treatment, and cell cryopreservation efficiency and cell recovery survival rate are greatly improved, immunocyte, stem cell based on the preparation of this frozen stock solution etc. have broad prospect of application in disease treatment, biological beauty etc..
Description
Technical field
The present invention relates to cell therapy and field of medicaments, specifically, is related to a kind of cells frozen storing liquid and preparation method thereof
With application.
Background technology
Cell therapy refers to the human body cell transplanting for transforming normal or bioengineering or input patient's body, new input
Cell can substitute damaged cell or there is stronger immunologic cytotoxicity function, so as to reach treatment disease purpose.Cell
Treatment is shown in treating cancer, blood disease, cardiovascular disease, diabetes, senile dementia etc. higher and higher applies valency
Value.In general, cell therapy includes immune cell therapy and the major class of stem-cell therapy two.Common immune cell therapy species
Including NK cells, CIK cell, DC-CIK cells, til cell, LAK cells, CAR-T cells etc..Common stem-cell therapy kind
Class include embryonic stem cell, iPS cells, fat mesenchymal stem cell, mesenchymal stem cells MSCs, umbilical cord mesenchymal stem cells,
Umbilical cord blood mesenchymal stem cellses, deciduous teeth mescenchymal stem cell, amnion mesenchymal stem cell, placenta mesenchyma stem cell etc..
In cell therapy clinic, often need to preserve cell liquid nitrogen frozen, be used further to continue after a period of time culture or
Clinical direct feedback.At present, the conventional cells frozen storing liquid in laboratory or the cells frozen storing liquid of commercialization often add foreign sera or
Foreign protei, in cell therapy in the presence of the risk for introducing pollution or anaphylactogen.In addition, these cells frozen storing liquids are also present at present
After cell recovery the shortcomings of poor activity.In a word, clinically actual demand be present to efficient, safe cells frozen storing liquid at present.
The content of the invention
Cell cryopreservation efficiency can be effectively improved it is an object of the invention to provide one kind and improves cell recovery survival rate, can be pacified
It is complete to be used for clinical cells frozen storing liquid and preparation method and application.
In order to realize the object of the invention, cells frozen storing liquid of the invention, by containing human serum albumins, dimethyl sulfoxide (DMSO) and
The cell culture medium composition of high polymer material；In the culture medium percentage by volume of human serum albumins be 1-5% (preferably
4%), the percentage by volume of dimethyl sulfoxide (DMSO) is 5-15% (preferably 10%), and the mass percent of high polymer material is 0.1-
10% (preferably 5%).Preferably, the percentage by volume of human serum albumins is 4% in the culture medium, the body of dimethyl sulfoxide (DMSO)
Product percentage is 10%, and the mass percent of high polymer material is 5%.
In the present invention, the high polymer material is that main chain is hydrocarbon chain, and side base is total to for the macromolecule of carboxyl and hydrophobic grouping
Polymers.The mass percent of carboxyl is 0.01%-40% in the high polymer material.The number-average molecular weight of the high polymer material
For 3,000-100,000.The hydrophobic grouping is selected from least one of alkyl, phenyl, ester group etc..
Preferably, the high polymer material is selected from methacrylic acid-methacrylate copolymer, styrene-methyl third
Olefin(e) acid copolymer, acrylic acid and acrylic ester copolymers, Poly(D,L-lactide-co-glycolide, alkyl maleates-maleic acid are common
At least one of polymers, styrene-methacrylate-acrylic acid triblock copolymer etc..
It is highly preferred that the high polymer material is selected from methacrylic acid-methacrylate copolymer, polylactic acid-glycolic base
At least one of acetate multipolymer and styrene-t block copolymer etc..
The cells frozen storing liquid of the present invention can be prepared as follows obtaining：
(1) high polymer material is dissolved in dimethyl sulfoxide (DMSO), obtains solution I；
(2) human serum albumins is added into solution I, gained mixture is added in cell culture medium, obtains cell
Frozen stock solution.
The present invention also provides application of the cells frozen storing liquid in cell cryopreservation and recovery.
It is thin that the cell includes but is not limited to NK cells, CIK cell, DC-CIK cells, til cell, LAK cells, CART
Born of the same parents, mammalian embryonic stem cell, iPS cells, fat mesenchymal stem cell, mesenchymal stem cells MSCs, umbilical cord mesenchyma are done
Cell, umbilical cord blood mesenchymal stem cellses, deciduous teeth mescenchymal stem cell, amnion mesenchymal stem cell or placenta mesenchyma stem cell.
Foregoing application, the specific method of Cell Cryopreservation are：The cells frozen storing liquid is added into cell, is obtained thin
Born of the same parents' suspension (concentration 1 × 105~1 × 106Individual/mL)；Then cell suspending liquid program is cooled to -80 DEG C, is transferred to liquid nitrogen guarantor
Deposit.
Preferably, described program cooling refers to, with 5 DEG C/h of cooling rate, -80 DEG C are down to by room temperature.
The present invention further provides the kit containing described cells frozen storing liquid.
The cells frozen storing liquid is preparing the cells such as immunocyte, stem cell in clinical disease treatment, biological beauty
Application, fall within protection scope of the present invention.
Compared with prior art, the present invention has advantages below：
(1) biological safety is high.The cells frozen storing liquid of the present invention is by human serum albumins, dimethyl sulfoxide (DMSO), macromolecule material
Material and culture medium are formed, and foreign protei or serum are free of in cells frozen storing liquid, substantially increase the security of clinical cytology treatment.
(2) cell cryopreservation efficiency and cell recovery survival rate are greatly improved.Compared with conventional cell frozen stock solution, the present invention
Cells frozen storing liquid can greatly improve cell, especially stem cell, the cell recovery survival rate of immunocyte, maximum lift amplitude
Up to 67%.
(3) using the immunocyte of cells frozen storing liquid preparation of the invention, stem cell etc. in disease treatment, biological beauty
Etc. have broad application prospects.
Brief description of the drawings
Fig. 1 is that cells frozen storing liquid influences to contrast on cell survival rate in the embodiment of the present invention 1.
Fig. 2 is that polymer concentration influences on cell survival rate in the embodiment of the present invention 2.
Fig. 3 is that different people seralbumin concentration influences on cell survival rate in the embodiment of the present invention 3.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
The percentage sign " % " being related in the present invention, if not specified, refers to mass percent；But the percentage of solution
Than unless otherwise specified, referring to the grams containing solute in 100mL solution.
The preparation method of cells frozen storing liquid is as follows in following examples：
(1) high polymer material is dissolved in dimethyl sulfoxide (DMSO), obtains solution I；
(2) human serum albumins is added into solution I, gained mixture is added in cell culture medium, obtains cell
Frozen stock solution.
1 two kinds of different cells frozen storing liquids of embodiment compare
Cell：Human umbilical cord mesenchymal stem cells.
Cryopreservation methods：The cells frozen storing liquid is added into cell, obtains cell suspending liquid (concentration 1 × 105~1 × 106
Individual/mL)；Then cell suspending liquid is cooled to -80 DEG C with 5 DEG C/h of cooling rate programs, is transferred to Liquid nitrogen storage.
Cell recovery method：Using conventional method recovery cell.
Cells frozen storing liquid A：10%+10% dimethyl sulfoxide (DMSO) of hyclone+culture medium；
Cells frozen storing liquid B：+ 1% methacrylic acid of dimethyl sulfoxide (DMSO) of 0.1% human serum albumins+10%-methacrylic acid
Ester copolymer (Mw about 34,000, CAS：25086-15-1)+culture medium.
Cell survival rate contrast is as shown in Figure 1 after cryopreservation.It will be seen from figure 1 that frozen using cells frozen storing liquid A
Cell is deposited, cell survival rate 82% after recovery；Using cells frozen storing liquid B freeze-stored cells, cell survival rate 93% after recovery.
The polymer concentration of embodiment 2 influences on cell survival rate
Cell：Human umbilical cord mesenchymal stem cells.
Cells frozen storing liquid：Dimethyl sulfoxide (DMSO)+0.1-10% the methacrylic acids of 5% human serum albumins+10%-metering system
Acid ester copolymer (Mw about 34,000, CAS：25086-15-1)+culture medium.Wherein, methacrylic acid-methacrylate copolymers
The concentration of thing is respectively 0.2%, 0.8%, 1.0%, 1.5%, 5.0% and 10.0%.
Cell survival rate is shown in Fig. 2 after cryopreservation, and methacrylic acid-methacrylate is common as can be seen from Figure 2
When the concentration of polymers is respectively 0.2%, 0.8%, 1.0%, 1.5%, 5.0% and 10.0%, cellular activities after corresponding recovery
Rate is respectively 40%, 50%, 75%, 80%, 95% and 81%.
The different people seralbumin concentration of embodiment 3 influences on cell survival rate
Cell：Human umbilical cord mesenchymal stem cells.
Cells frozen storing liquid：+ 3% methacrylic acid of dimethyl sulfoxide (DMSO) of 1-5% human serum albumins+10%-methacrylic acid
Ester copolymer (Mw about 34,000, CAS：25086-15-1)+culture medium.Wherein, the concentration of human serum albumins be respectively 1%,
2%th, 3%, 4% and 5%.
Cell survival rate is shown in Fig. 3 after cryopreservation, and the concentration of human serum albumins is respectively as can be seen from Figure 3
1%th, 2%, 3%, 4% and 5% when, cell survival rate is respectively 85%, 90%, 92%, 95% and 94% after corresponding recovery.
The Poly(D,L-lactide-co-glycolide of embodiment 4 3% freezes effect
Cell：Human umbilical cord mesenchymal stem cells.
Cells frozen storing liquid：+ 3% Poly(D,L-lactide-co-glycolide of dimethyl sulfoxide (DMSO) of 5% human serum albumins+10%+training
Support base.
Cell survival rate is 94 ± 3% after cryopreservation.
The Poly(D,L-lactide-co-glycolide of embodiment 5 1% freezes effect
Cell：Human umbilical cord mesenchymal stem cells.
Cells frozen storing liquid：+ 1% Poly(D,L-lactide-co-glycolide of dimethyl sulfoxide (DMSO) of 5% human serum albumins+10%+training
Support base.
Cell survival rate is 91 ± 3% after cryopreservation.
Embodiment 6HeLa cells and L929 cells freeze effect
Cell：HeLa and L929.
Cells frozen storing liquid：+ 5% methacrylic acid of dimethyl sulfoxide (DMSO) of 5% human serum albumins+10%-methacrylate
Copolymer (Mw about 34,000, CAS：25086-15-1)+DMEM culture mediums.
HeLa cells and L929 cells are frozen one week with frozen stock solution.Then cell recovery, trypan blue, which counts, calculates cell
Survival rate.HeLa cells and L929 cell recovery survival rates are respectively 97 ± 3%, 96 ± 4%.
Embodiment 7DC-CIK cells freeze effect
Cell：DC-CIK cells.
Cells frozen storing liquid：+ 3% methacrylic acid of dimethyl sulfoxide (DMSO) of 5% human serum albumins+10%-methacrylate
Copolymer (Mw about 34,000, CAS：25086-15-1)+DMEM culture mediums.
The preparation method of PMNC is as follows：Liquid density level bands is layered according to the Ficoll-Hypaque of routine
The PMNC of centrifugal process separation patient is spent, to be put into after 75% alcohol wipe blood bag in Biohazard Safety Equipment；With iodine
Wine and 75% alcohol disinfecting blood bag inject the mouth of pipe 2 times, extract blood out with 50ml syringes, are transferred to 50ml centrifuge tubes, often pipe 20ml
Left and right；After blood pumps, 20ml RPMI1640 are added in blood bag, reverse blood bag up and down 2-3 times, blood bag is cleaned, will clean
Liquid is transferred to 50ml centrifuge tubes；Often pipe peripheral blood adds 20mlRPMI1640 to about 40ml, is mixed peripheral blood with 10ml pipettes
It is even；8min is centrifuged with rotating speed 320g；Supernatant is gently suctioned out into about 17ml, discards, again breaks up precipitation；Cell is counted, will be thin
Born of the same parents' concentration is adjusted to 1.5 × 109-2×109/22ml；Take in the new 50ml centrifuge tubes of 12.5ml ficoll to one, by 22ml peripheries
Blood cell suspension is added slowly on separating liquid along tube wall, and pays attention to keeping both interfaces clear；With rotating speed 450g at room temperature
Centrifugation 25 minutes, can clearly be divided into four layers in pipe；With the milky mononuclearcell layer of the careful absorption of pipette, and it is transferred to
Into new 50ml centrifuge tubes, with the PBS pipette more than or equal to mononuclearcell layer volume；It is transferred to centrifuge tube
In, centrifuged 10 minutes with rotating speed 450g room temperatures, remove the blood platelet mixed.
The preparation method of lymphocyte is as follows：Pass through the lymphocyte and adherent mononuclear cells in PMNC
Performance difference is separated, and lymphocyte is suspension growth in nutrient solution, and monocyte can be adsorbed onto the bottom of culture vessel.
Mononuclearcell is precipitated and is resuspended with AIM-V (serum free medium), with 7 × 107The density of/6ml/ disks is inoculated in 10cm cultures
Ware；In 37 DEG C of 5%CO2Cultivated 2 hours in the incubator of concentration；Upper strata suspension cell is transferred to 50ml centrifuge tubes with pipette
In, wash culture dish twice with 3ml RPMI1640, the lymphocyte of suspension is all accepted as far as possible, is transferred in 50ml centrifuge tubes；
Centrifuged 7 minutes with rotating speed 320g room temperatures, shift supernatant, be resuspended with AIM-V culture mediums, pipette is blown and beaten several times back and forth, by cell
Suspension mixes, and with above method cell count, calculates cell survival rate；Equally with 7 × 107The density of/6ml/ disks connects again
Kind as far as possible rejects the monocyte in lymphocyte in 10cm culture dishes.
The preparation method of antigen presenting cell-BMDC is as follows：First time is collected to the 10cm after lymphocyte
Culture dish (is pasted with monocyte), and often disk adds AIM-V nutrient solutions 3ml cleaning once, removes lymphocyte nuclear and residual as far as possible
The RPMI1640 nutrient solutions stayed, each culture dish addition 3ml AIM-V nutrient solutions, add the Dendritic Cells Induced factor afterwards
GM-CSF (50ng/ml) and IL-4 (25ng/ml), in 37 DEG C of 5%CO218-48 hours are cultivated in incubator；With 10ml pipettes
The suspension cell in monocyte culture dish is transferred in 50ml centrifuge tubes, and with 3ml PBS culture dishes；Add 3ml
5mM EDTA/PBS, foster 5-10min in incubator；Culture dish is transferred to Biohazard Safety Equipment by culture dish from incubator
In, adherent dendritic cells are swept away with original 3ml EDTA/PBS, are transferred in 50ml centrifuge tubes；With rotating speed 320g room temperatures from
Heart 7min；Supernatant is removed, is suspended with cells frozen storing liquid, with 1 × 108/ 2ml concentration is transferred to cryopreservation tube, and lid is twisted, drop of loading program
Wen Yi, place more than 4 hours, be transferred to DC-CIK cell cryopreservations one week in liquid nitrogen in -80 DEG C of refrigerators.Then cell recovery,
Trypan blue, which counts, calculates cell survival rate.DC-CIK cell recovery survival rates are respectively 95 ± 3%.
Embodiment 8DC-CIK cells freeze effect
Cell：DC-CIK cells.
Cells frozen storing liquid：+ 5% methacrylic acid of dimethyl sulfoxide (DMSO) of 5% human serum albumins+10%-methacrylate
Copolymer (Mw about 34,000, CAS：25086-15-1)+DMEM culture mediums.
The preparation method of PMNC is as follows：Liquid density level bands is layered according to the Ficoll-Hypaque of routine
The PMNC of centrifugal process separation patient is spent, to be put into after 75% alcohol wipe blood bag in Biohazard Safety Equipment；With iodine
Wine and 75% alcohol disinfecting blood bag inject the mouth of pipe 2 times, extract blood out with 50ml syringes, are transferred to 50ml centrifuge tubes, often pipe 20ml
Left and right；After blood pumps, 20ml RPMI1640 are added in blood bag, reverse blood bag up and down 2-3 times, blood bag is cleaned, will clean
Liquid is transferred to 50ml centrifuge tubes；Often pipe peripheral blood adds 20ml RPMI1640 to about 40ml, with 10ml pipettes by peripheral blood
Mix；8min is centrifuged with rotating speed 320g；Supernatant is gently suctioned out into 17ml or so, discards, again breaks up precipitation；Number cell, will
Cell concentration is adjusted to 1.5 × 109-2×109/22ml；Take in the new 50ml centrifuge tubes of 12.5ml ficoll to one, by 22ml
Peripheral blood cells suspension is added slowly on separating liquid along tube wall, and pays attention to keeping both interfaces clear；With rotating speed 450g room temperatures
Centrifugation 25 minutes, can clearly be divided into four layers in pipe；With the milky mononuclearcell layer of the careful absorption of pipette, and it is transferred to
Into new 50ml centrifuge tubes, with the PBS pipette than or equal to mononuclearcell layer volume；It is transferred to centrifuge tube
In, centrifuged 10 minutes with rotating speed 450g room temperatures, remove the blood platelet mixed.
The preparation method of lymphocyte is as follows：By obtaining lymphocyte and adherent mononuclear cells in PMNC
Performance difference is separated, and lymphocyte is suspension growth in nutrient solution, and monocyte can be adsorbed onto the bottom of culture vessel.
Mononuclearcell is precipitated and is resuspended with AIM-V (serum free medium), with 7 × 107The density of/6ml/ disks is inoculated in 10cm cultures
Ware；In 37 DEG C of 5%CO2Cultivated 2 hours in the incubator of concentration；By the suspension cell on upper strata with pipette be transferred to 50ml from
In heart pipe, culture dish is washed twice with 3ml RPMI1640, the lymphocyte of suspension is all accepted as far as possible, is transferred to 50ml centrifuge tubes
In；Centrifuged 7 minutes with rotating speed 320g room temperatures, shift supernatant, be resuspended with AIM-V culture mediums, pipette is blown and beaten several times back and forth, will be thin
Born of the same parents' suspension mixes, and with above method cell count, calculates cell survival rate；Equally with 7 × 107The density of/6ml/ disks is again
10cm culture dishes are inoculated in, as far as possible reject the monocyte in lymphocyte.
The preparation method of antigen presenting cell-BMDC is as follows：First time is collected to the 10cm after lymphocyte
Culture dish (is pasted with monocyte), and often disk adds AIM-V nutrient solutions 3ml cleaning once, removes lymphocyte nuclear and residual as far as possible
RPMI1640 nutrient solutions are stayed, each culture dish adds 3ml AIM-V nutrient solutions afterwards, adds Dendritic Cells Induced factor GM-
CSF (50ng/ml) and IL-4 (25ng/ml), in 37 DEG C of 5%CO218-48 hours are cultivated in the incubator of concentration；Moved with 10ml
Suspension cell in monocyte culture dish is transferred in 50ml centrifuge tubes by liquid pipe, and with 3ml PBS culture dishes；Add
Enter 3ml 5mM EDTA/PBS, 5-10min is fostered in incubator；Culture dish is transferred to biology by culture dish from incubator
In safety cabinet, adherent dendritic cells are swept away with original 3ml EDTA/PBS, are transferred in 50ml centrifuge tubes；With rotating speed 320g
Room temperature centrifuges 7min；Supernatant is removed, is suspended with cells frozen storing liquid, with 1 × 108/ 2ml concentration is transferred to cryopreservation tube, and lid is twisted, and loads
Programmed cooling instrument, is placed on -80 DEG C of refrigerators, places more than 4 hours, is transferred to DC-CIK cell cryopreservations one week in liquid nitrogen.Then
Cell recovery, trypan blue, which counts, calculates cell survival rate.
Cell is resuspended with RPMI1640, Trypan Blue, carries out cell count and calculates survival rate.DC-CIK cell recoveries
Survival rate is respectively 92 ± 3%.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of cells frozen storing liquid, it is characterised in that by containing the thin of human serum albumins, dimethyl sulfoxide (DMSO) and high polymer material
Born of the same parents' culture medium forms；The percentage by volume of human serum albumins is 1-5% in the culture medium, the volume basis of dimethyl sulfoxide (DMSO)
Number is 5-15%, and the mass percent of high polymer material is 0.1-10%；
Wherein, the high polymer material is that main chain is hydrocarbon chain, and side base is the high-molecular copolymer of carboxyl and hydrophobic grouping；It is described
The mass percent of carboxyl is 0.01%-40% in high polymer material, and the number-average molecular weight of the high polymer material is 3,000-
100,000, the hydrophobic grouping is selected from least one of alkyl, phenyl, ester group.
2. cells frozen storing liquid according to claim 1, it is characterised in that the high polymer material be selected from methacrylic acid-
Methacrylate copolymer, Styrene-methyl Acrylic Acid Copolymer, acrylic acid and acrylic ester copolymers, polylactic acid-glycolic base
In acetate multipolymer, alkyl maleates-maleic acid, styrene-methacrylate-acrylic acid triblock copolymer
At least one.
3. cells frozen storing liquid according to claim 2, it is characterised in that the high polymer material be selected from methacrylic acid-
In methacrylate copolymer, Poly(D,L-lactide-co-glycolide and styrene-t block copolymer at least
It is a kind of.
4. according to the cells frozen storing liquid described in claim any one of 1-3, it is characterised in that human serum in the cell culture medium
The percentage by volume of albumin is 4%, and the percentage by volume of dimethyl sulfoxide (DMSO) is 10%, and the mass percent of high polymer material is
5%.
5. any one of the claim 1-4 cell cryopreservation liquid and preparation method thereofs, it is characterised in that comprise the following steps：
(1) high polymer material is dissolved in dimethyl sulfoxide (DMSO), obtains solution I；
(2) human serum albumins is added into solution I, gained mixture is added in cell culture medium, obtains cell cryopreservation
Liquid.
6. application of any one of the claim 1-4 cells frozen storing liquids in cell cryopreservation and recovery.
7. application according to claim 6, it is characterised in that the cell be NK cells, CIK cell, DC-CIK cells,
Filled between til cell, LAK cells, CART cells, mammalian embryonic stem cell, iPS cells, fat mesenchymal stem cell, marrow
Matter stem cell, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cellses, deciduous teeth mescenchymal stem cell, amnion mesenchymal stem cell
Or placenta mesenchyma stem cell.
8. the application according to claim 6 or 7, it is characterised in that the specific method of Cell Cryopreservation is：Into cell
The cells frozen storing liquid is added, obtains concentration as 1 × 105~1 × 106Individual/mL cell suspending liquid；Then by cell suspending liquid
Program is cooled to -80 DEG C, is transferred to Liquid nitrogen storage.
9. application according to claim 8, it is characterised in that described program cooling refers to the cooling speed with 5 DEG C/h
Degree, -80 DEG C are down to by room temperature.
10. the kit containing the cells frozen storing liquid described in claim any one of 1-4.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108094410A (en) * | 2018-01-26 | 2018-06-01 | 山东大学齐鲁医院 | A kind of skin profound hypothermia protective agent and vitrification method |
| CN110468095A (en) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | A kind of renal epithelial cell and its preparation method and application |
| WO2021213446A1 (en) * | 2020-04-21 | 2021-10-28 | 佧珐药业有限公司 | Low-temperature storage of biological samples |
| CN115606583A (en) * | 2022-10-26 | 2023-01-17 | 河北生命原点生物科技有限公司 | Cryopreservation protection solution for umbilical cord stem cells, and preparation method and application thereof |
| CN115644168A (en) * | 2022-11-09 | 2023-01-31 | 湖南开启时代生物科技有限责任公司 | Frozen stock solution of finished immune cells and preparation method and freezing method thereof |
Citations (1)
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| CN106520679A (en) * | 2016-11-30 | 2017-03-22 | 张晓南 | Kit for culturing pancreas stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106520679A (en) * | 2016-11-30 | 2017-03-22 | 张晓南 | Kit for culturing pancreas stem cells |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108094410A (en) * | 2018-01-26 | 2018-06-01 | 山东大学齐鲁医院 | A kind of skin profound hypothermia protective agent and vitrification method |
| CN108094410B (en) * | 2018-01-26 | 2021-08-31 | 山东大学齐鲁医院 | Skin deep hypothermia protective agent and skin preservation method |
| CN110468095A (en) * | 2019-09-05 | 2019-11-19 | 安徽中盛溯源生物科技有限公司 | A kind of renal epithelial cell and its preparation method and application |
| WO2021213446A1 (en) * | 2020-04-21 | 2021-10-28 | 佧珐药业有限公司 | Low-temperature storage of biological samples |
| CN115606583A (en) * | 2022-10-26 | 2023-01-17 | 河北生命原点生物科技有限公司 | Cryopreservation protection solution for umbilical cord stem cells, and preparation method and application thereof |
| CN115606583B (en) * | 2022-10-26 | 2023-11-10 | 河北生命原点生物科技有限公司 | Cryopreservation protection liquid for umbilical cord stem cells, and preparation method and application thereof |
| CN115644168A (en) * | 2022-11-09 | 2023-01-31 | 湖南开启时代生物科技有限责任公司 | Frozen stock solution of finished immune cells and preparation method and freezing method thereof |
| CN115644168B (en) * | 2022-11-09 | 2024-03-01 | 湖南开启时代生物科技有限责任公司 | Frozen stock solution of finished immune cells, preparation method thereof and frozen stock method |
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