CN114540291A - Umbilical cord mesenchymal stem cell resuscitation protection solution and resuscitation method - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 21
- 210000004027 cell Anatomy 0.000 claims abstract description 38
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 32
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 32
- CLAHOZSYMRNIPY-UHFFFAOYSA-N 2-hydroxyethylurea Chemical compound NC(=O)NCCO CLAHOZSYMRNIPY-UHFFFAOYSA-N 0.000 claims abstract description 23
- BUQLXKSONWUQAC-UHFFFAOYSA-N Parthenolide Natural products CC1C2OC(=O)C(=C)C2CCC(=C/CCC1(C)O)C BUQLXKSONWUQAC-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229940031575 hydroxyethyl urea Drugs 0.000 claims abstract description 23
- KTEXNACQROZXEV-PVLRGYAZSA-N parthenolide Chemical compound C1CC(/C)=C/CC[C@@]2(C)O[C@@H]2[C@H]2OC(=O)C(=C)[C@@H]21 KTEXNACQROZXEV-PVLRGYAZSA-N 0.000 claims abstract description 23
- 229940069510 parthenolide Drugs 0.000 claims abstract description 23
- 239000001963 growth medium Substances 0.000 claims abstract description 22
- 230000001681 protective effect Effects 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims description 45
- 238000010257 thawing Methods 0.000 claims description 10
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
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- 239000007640 basal medium Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 15
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- 238000007710 freezing Methods 0.000 abstract description 2
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- 230000002195 synergetic effect Effects 0.000 abstract 1
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- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
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- 210000000663 muscle cell Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
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Abstract
The invention discloses a resuscitation protection solution for umbilical cord mesenchymal stem cells, which comprises a basic culture medium and the following components added in the basic culture medium: parthenolide, hydroxyethyl urea and polyethylene glycol. The umbilical cord mesenchymal stem cells after being frozen are firstly subjected to recovery culture for 24h by using the recovery protective solution, so that the survival rate and the proliferation activity of the umbilical cord mesenchymal stem cells are improved, and the adverse effects of ultra-low temperature freezing of liquid nitrogen on the umbilical cord mesenchymal stem cells are reduced due to the synergistic effect of parthenolide, hydroxyethyl urea and polyethylene glycol in the recovery protective solution. The invention also provides a recovery method of the umbilical cord mesenchymal stem cells, and the recovery protection solution provided by the invention is adopted to effectively improve the recovery efficiency of the umbilical cord mesenchymal stem cells, so that the cells have higher activity and are used for clinical research and the like.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a recovery protective solution and a recovery method for umbilical cord mesenchymal stem cells.
Background
The mesenchymal stem cells are adult stem cells derived from early mesoderm, have high self-renewal capacity and multidirectional differentiation potential, can proliferate and differentiate to various tissues such as bone, tendon, muscle, fat and stromal cells, have weak immunogenicity, and are a commonly used seed cell source in tissue engineering.
The umbilical cord mesenchymal stem cells are derived from umbilical cord. The umbilical cord is the waste after the delivery of the fetus, has wide sources and does not have ethical problems. The cell has lower immunogenicity, stronger original, proliferation and differentiation capacities, can still maintain the characteristics of the stem cells after continuous and repeated passages, can provide sufficient cell sources for scientific research and clinic, and has wide application prospect.
And (4) if the umbilical cord mesenchymal stem cells obtained by separation culture are not timely, selecting proper conditions for storage. Cryopreservation of umbilical cord mesenchymal stem cells in liquid nitrogen has become a conventional technical means for prolonging the preservation time of cells. The cell frozen by liquid nitrogen needs to be subjected to a recovery process to recover normal activity, the common recovery process is to melt a frozen tube in a water bath, add a culture medium for dilution, then centrifugally remove supernatant, and add the culture medium again for heavy suspension to perform normal amplification culture. Although the recovery process is carried out, the activity of the umbilical cord mesenchymal stem cells is obviously reduced compared with normal cells which are not frozen, the cell survival rate and the proliferation activity are not as good as those of the normal cells which are not frozen, the cell survival rate and the proliferation activity are sometimes greatly reduced, and the subsequent clinical application effect of the recovered cells cannot be ensured. Therefore, there is a need to provide a resuscitation protection solution for umbilical cord mesenchymal stem cells, which is used in the process of umbilical cord mesenchymal stem cell resuscitation and can improve cell resuscitation efficiency.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the umbilical cord mesenchymal stem cell resuscitation protection solution which is simple in components and can effectively improve the proliferation activity and differentiation capacity of cells after cryopreservation and resuscitation.
The invention also aims to provide a recovery method of umbilical cord mesenchymal stem cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
a resuscitation protection solution for umbilical cord mesenchymal stem cells comprises a basic culture medium and the following components added in the basic culture medium: parthenolide, hydroxyethyl urea and polyethylene glycol.
Preferably, the basal medium is DMEM medium.
Preferably, the final concentrations of parthenolide, hydroxyethyl urea, polyethylene glycol in the culture medium are: 9.6-14.5ng/mL of parthenolide, 17.5-23.5ng/mL of hydroxyethyl urea and 7.2-10.5 mu g/mL of polyethylene glycol.
Preferably, the final concentrations of parthenolide, hydroxyethyl urea, polyethylene glycol in the culture medium are: 11.5ng/mL of parthenolide, 20ng/mL of hydroxyethyl urea and 8.7 mu g/mL of polyethylene glycol.
Preferably, the polyethylene glycol has an average molecular weight of 2000.
The second purpose of the invention is realized by adopting the following technical scheme:
a method for recovering umbilical cord mesenchymal stem cells is characterized in that the recovery protective solution is added in the recovery process.
Preferably, the method for recovering umbilical cord mesenchymal stem cells comprises the following processes: thawing frozen umbilical cord mesenchymal stem cells in water bath at 37-42 deg.C for thawing, adding resuscitation protecting solution for dilution, centrifuging to collect cells, resuspending the collected cells with resuscitation protecting solution, and 5% CO at 37 deg.C2Standing for 24h to finish.
Preferably, the cell density after resuspension with the resuscitation protection solution is 2-5X 104one/mL.
Compared with the prior art, the invention has the beneficial effects that: the umbilical cord mesenchymal stem cell recovery protective solution is prepared by adding parthenolide, hydroxyethyl urea and polyethylene glycol into a basic culture medium, and the umbilical cord mesenchymal stem cells subjected to cryopreservation are recovered and cultured for 24 hours by using the recovery protective solution, so that the survival rate and the proliferation activity of the umbilical cord mesenchymal stem cells are improved, and the adverse effect of ultralow-temperature cryopreservation of liquid nitrogen on the umbilical cord mesenchymal stem cells is reduced.
The invention also provides a recovery method of the umbilical cord mesenchymal stem cells, and the recovery protection solution provided by the invention is adopted to effectively improve the recovery efficiency of the umbilical cord mesenchymal stem cells, so that the umbilical cord mesenchymal stem cells have higher activity and are used for clinical research and the like.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Preparing umbilical cord mesenchymal stem cells: cleaning umbilical cord tissue with PBS, removing venous and arterial blood vessels, peeling off Fahrenheit glue tissue, and cutting into 1-2mm pieces3Tissue blocks of size were seeded in Petri dishes at 37 ℃ in DMEM/F12 medium with 10% FBS and 5% CO2Culture of (2)Culturing in a box, digesting with 0.25% trypsin for subculture when the cell fusion degree reaches 85%, wherein the subculture ratio is 1:3, freezing and storing P3 generation cells by a conventional method for 1 month, and taking out for resuscitation experiment.
Example 1
A resuscitation protection solution for umbilical mesenchymal stem cells comprises a DMEM culture medium and the following components added in the culture medium: parthenolide, hydroxyethyl urea and polyethylene glycol with the molecular weight of 2000, wherein the final concentration of each component in the culture medium is as follows: 9.6ng/mL of parthenolide, 17.5ng/mL of hydroxyethyl urea and 7.2 mu g/mL of polyethylene glycol.
A method for resuscitating umbilical cord mesenchymal stem cells comprises the following steps: taking P3 generation umbilical cord mesenchymal stem cells frozen in liquid nitrogen for 1 month, thawing and thawing the frozen tube in water bath at 37 ℃, adding resuscitation protection solution with 5 times volume for dilution, centrifuging at 1000rpm for 5min, removing supernatant, collecting umbilical cord mesenchymal stem cells, resuspending the collected cells in a resuscitation protection solution, inoculating the resuscitative protection solution into a 12-hole plate with 2mL per hole, wherein the cell density in the resuscitation protection solution is 2 multiplied by 104one/mL, 5% CO at 37 ℃2The culture box is kept still for 24 hours, and the operation is finished.
Example 2
A resuscitation protection solution for umbilical cord mesenchymal stem cells comprises a DMEM culture medium and the following components added in the culture medium: parthenolide, hydroxyethyl urea and polyethylene glycol with the molecular weight of 2000, wherein the final concentration of each component in the culture medium is as follows: 11.5ng/mL of parthenolide, 20ng/mL of hydroxyethyl urea and 8.7 mu g/mL of polyethylene glycol.
A method for resuscitating umbilical cord mesenchymal stem cells comprises the following steps: taking P3 generation umbilical cord mesenchymal stem cells frozen in liquid nitrogen for 1 month, thawing and thawing the frozen tube in water bath at 40 ℃, adding resuscitation protection solution with 4 times volume for dilution, centrifuging at 1000rpm for 5min, removing supernatant, collecting umbilical cord mesenchymal stem cells, resuspending the collected cells in a resuscitation protection solution, inoculating the resuscitative protection solution into a 12-hole plate with 2mL per hole, wherein the cell density in the resuscitation protection solution is 3 multiplied by 104one/mL, 5% CO at 37 ℃2The culture box is kept still for 24 hours, and the operation is finished.
Example 3
A resuscitation protection solution for umbilical cord mesenchymal stem cells comprises a DMEM culture medium and the following components added in the culture medium: parthenolide, hydroxyethyl urea and polyethylene glycol with the molecular weight of 2000, wherein the final concentration of each component in the culture medium is as follows: 14.5ng/mL of parthenolide, 23.5ng/mL of hydroxyethyl urea and 10.5 mu g/mL of polyethylene glycol.
A method for resuscitating umbilical cord mesenchymal stem cells comprises the following steps: taking P3 generation umbilical cord mesenchymal stem cells frozen in liquid nitrogen for 1 month, thawing and thawing the frozen tube in water bath at 42 ℃, adding resuscitation protection solution with 3 times volume for dilution, centrifuging at 1000rpm for 5min, removing supernatant, collecting umbilical cord mesenchymal stem cells, resuspending the collected cells in a resuscitation protection solution, inoculating the resuscitated cells in a 12-hole plate with 2mL per hole, wherein the cell density in the resuscitation protection solution is 5 multiplied by 104one/mL, 5% CO at 37 ℃2The culture box is kept still for 24 hours, and the operation is finished.
Comparative example 1
Comparative example 1 provides a protective solution for umbilical cord mesenchymal stem cell resuscitation, which is different from example 1 in that: the same procedure as in example 1 was repeated except that parthenolide, hydroxyethyl urea and polyethylene glycol were omitted.
Comparative example 2
Comparative example 2 provides a protective solution for umbilical cord mesenchymal stem cell resuscitation, which is different from example 1 in that: parthenolide was omitted and the procedure was as in example 1.
Comparative example 3
Comparative example 3 provides a protective solution for umbilical cord mesenchymal stem cell resuscitation, which is different from example 1 in that: the hydroxyethyl urea was omitted and the procedure was as in example 1.
Comparative example 4
Comparative example 4 provides a protective solution for umbilical cord mesenchymal stem cell resuscitation, which is different from example 1 in that: the polyethylene glycol was omitted and the procedure was as in example 1.
Comparative example 5
Comparative example 5 provides a resuscitation protection solution for umbilical cord mesenchymal stem cells, which is different from example 1 in that: the polyethylene glycol was adjusted to polyethylene glycol having an average molecular weight of 1500, and the rest was the same as in example 1.
Comparative example 6
Comparative example 6 provides a protective solution for umbilical cord mesenchymal stem cell resuscitation, which is different from example 1 in that: the polyethylene glycol was adjusted to polyethylene glycol having an average molecular weight of 2500, and the rest was the same as in example 1.
Counting the recovery rate of the cells in examples 1 to 3 and comparative examples 1 to 6: after 24 hours of standing, 20. mu.L of the cell mixture and 20. mu.L of trypan blue solution were mixed and stained, and the number of live cells and dead cells was counted to calculate the cell viability, and the results are shown in Table 1.
TABLE 1
It can be seen from table 1 that the viability of umbilical cord mesenchymal stem cells is higher in examples 1 to 3 than in comparative examples 1 to 6. The compositions of the resuscitation protection solutions were adjusted in comparative examples 1 to 6, and the survival rate of cells was lower than that of examples 1 to 3. In comparative example 1, parthenolide, hydroxyethyl urea and polyethylene glycol are omitted, cells are revived only by using a basal medium, and it can be seen from table 1 that the cell viability rate is far lower than that of example 1. One of parthenolide, hydroxyethyl urea and polyethylene glycol was omitted in comparative examples 2 to 4, respectively, and the molecular weight of polyethylene glycol was adjusted in comparative example 5 and comparative example 6, respectively, and the cell viability was higher than that of the group in which polyethylene glycol was omitted in comparative example 4, but the polyethylene glycol having an average molecular weight of 2000 used in the present invention was less effective than that of example 1.
The cells recovered in examples 1 to 3 and comparative examples 1 to 6 were centrifuged, the protective solution was removed, the unfrozen P3 generation cells were used as a control group, and the cells were resuspended in an alpha-MEM complete medium (10% FBS and 80U/ml streptomycin and 80U/ml penicillin were added to the medium) and inoculated into 6-well plates at 37 ℃ and 5% CO2Cultured in an incubator with a cell density of 1X 104Continuously culturing for 3 days, taking out 100 mu L of cell culture solution, adding CCK8 reagent, incubating for 1h in dark place, detecting the absorbance at 450nm, and calculating the cell doubling time according to the following calculation formula: DT = t × [ lg 2/(lgOD)t- lgOD0）]，ODtIs the absorbance value, OD, of 3d0The results are shown in Table 2 as absorbance values at the start of the culture.
TABLE 2
As can be seen from Table 2, the doubling times of the umbilical cord mesenchymal stem cells in examples 1 to 3 are not much different from those of the control group, but are all shorter than those of comparative examples 1 to 6, indicating that the umbilical cord mesenchymal stem cell proliferation activities in examples 1 to 3 are superior to those of comparative examples 1 to 6. The compositions of the resuscitation protection liquid are adjusted in the comparative examples 1 to 6, and the proliferation activities of the umbilical cord mesenchymal stem cells are reduced to different degrees, which shows that the resuscitation protection liquid provided by the invention can effectively improve the proliferation activity of the umbilical cord mesenchymal stem cells after cryopreservation.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (8)
1. The umbilical cord mesenchymal stem cell resuscitation protection solution is characterized by comprising a basic culture medium and the following components added in the basic culture medium: parthenolide, hydroxyethyl urea and polyethylene glycol.
2. The umbilical cord mesenchymal stem cell resuscitation protection solution according to claim 1, wherein the basal medium is a DMEM medium.
3. The umbilical mesenchymal stem cell resuscitation protection solution according to claim 1, wherein the final concentrations of parthenolide, hydroxyethyl urea and polyethylene glycol in the culture medium are as follows: 9.6-14.5ng/mL of parthenolide, 17.5-23.5ng/mL of hydroxyethyl urea and 7.2-10.5 mu g/mL of polyethylene glycol.
4. The umbilical cord mesenchymal stem cell resuscitation protection solution according to claim 1, wherein the final concentrations of parthenolide, hydroxyethyl urea and polyethylene glycol in the culture medium are as follows: 11.5ng/mL of parthenolide, 20ng/mL of hydroxyethyl urea and 8.7 mu g/mL of polyethylene glycol.
5. The umbilical cord mesenchymal stem cell resuscitation protection solution according to claim 1, wherein the polyethylene glycol has an average molecular weight of 2000.
6. A method for resuscitating umbilical cord mesenchymal stem cells, wherein the resuscitation protection solution of any one of claims 1 to 5 is added during the resuscitation process.
7. The method for resuscitating umbilical cord mesenchymal stem cells according to claim 6, comprising the following steps: thawing frozen umbilical cord mesenchymal stem cells in water bath at 37-42 deg.C for thawing, adding resuscitation protecting solution for dilution, centrifuging to collect cells, resuspending the collected cells with resuscitation protecting solution, and 5% CO at 37 deg.C2Standing for 24h to finish.
8. The method for resuscitating umbilical cord mesenchymal stem cells according to claim 7, wherein the cell density after resuspension with the resuscitating protective solution is 2-5 x 104one/mL.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107805626A (en) * | 2017-11-10 | 2018-03-16 | 武汉北度生物科技有限公司 | A kind of method for resuscitation of human umbilical cord mesenchymal stem cells |
| CN108812643A (en) * | 2018-07-18 | 2018-11-16 | 银丰生物工程集团有限公司 | Human placenia membrane tissue prepares cryopreservation methods and application |
| CN111838130A (en) * | 2020-05-28 | 2020-10-30 | 深圳华大基因细胞科技有限责任公司 | Protective solution and preparation method and application thereof |
| CN113136364A (en) * | 2021-04-13 | 2021-07-20 | 广东唯泰生物科技有限公司 | Preparation method and recovery method of decidua-muralis mesenchymal stem cells |
| CN113403272A (en) * | 2021-07-23 | 2021-09-17 | 廊坊康宝汇泰生物技术有限公司 | Culture medium of primary umbilical cord mesenchymal stem cells and application thereof |
-
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- 2022-02-23 CN CN202210165469.5A patent/CN114540291B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107805626A (en) * | 2017-11-10 | 2018-03-16 | 武汉北度生物科技有限公司 | A kind of method for resuscitation of human umbilical cord mesenchymal stem cells |
| CN108812643A (en) * | 2018-07-18 | 2018-11-16 | 银丰生物工程集团有限公司 | Human placenia membrane tissue prepares cryopreservation methods and application |
| CN111838130A (en) * | 2020-05-28 | 2020-10-30 | 深圳华大基因细胞科技有限责任公司 | Protective solution and preparation method and application thereof |
| CN113136364A (en) * | 2021-04-13 | 2021-07-20 | 广东唯泰生物科技有限公司 | Preparation method and recovery method of decidua-muralis mesenchymal stem cells |
| CN113403272A (en) * | 2021-07-23 | 2021-09-17 | 廊坊康宝汇泰生物技术有限公司 | Culture medium of primary umbilical cord mesenchymal stem cells and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| OLENA ROGULSKA 等: "DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival", vol. 69, pages 265 - 276, XP036197628, DOI: 10.1007/s10616-016-0055-2 * |
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