CN109628395A - A kind of digestion method improving the umbilical cord mesenchymal stem cells rate of recovery - Google Patents
A kind of digestion method improving the umbilical cord mesenchymal stem cells rate of recovery Download PDFInfo
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- CN109628395A CN109628395A CN201910145681.3A CN201910145681A CN109628395A CN 109628395 A CN109628395 A CN 109628395A CN 201910145681 A CN201910145681 A CN 201910145681A CN 109628395 A CN109628395 A CN 109628395A
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- umbilical cord
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- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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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
- C12N2510/00—Genetically modified cells
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Abstract
The invention discloses a kind of digestion methods for improving the umbilical cord mesenchymal stem cells rate of recovery, it is characterised in that: the following steps are included: the physiological saline containing ALB is added into the umbilical cord mesenchymal stem cells mixed liquor containing digestive pharmaceutical terminates pancreatin digestion.The beneficial effects of the present invention are: (1) present invention improves the rate of recovery of umbilical cord mesenchymal stem cells after digestion.(2) present invention reduces the toxigenic capacities of umbilical cord mesenchymal stem cells, are conducive to umbilical cord mesenchymal stem cells mass propgation, to meet the scientific research etc. of bigger demand.
Description
Technical field
The present invention relates to umbilical cord mesenchymal stem cells field more particularly to a kind of raising umbilical cord mesenchymal stem cells rate of recovery
Digestion method.
Background technique
Umbilical cord mesenchymal stem cells (Umbilical Cord Mesenchymal Stem Cells, UCMSCs) refer generally to
Lead to a kind of versatile stem cell of glue from neonatal umbilical cord Fahrenheit, UCMSCs can be with self-renewing, duplication and multiple directions
Broken up, before there is wide clinical application in terms of the diseases such as cartilage, muscle, ligament, nerve, liver, endothelium and cardiac muscle
Scape.
UCMSCs can generally disappear in separation and incubation by cutting navel cord, double enzymic digestions, adhere-wall culture, pancreatin
The operating process such as change and termination, collection and passage, and the groundwork that mescenchymal stem cell carries out after originally culture acquisition is just
It is passage and culture.Because of mescenchymal stem cell category adherent growth cell, during carrying out passage and training, need using
Pancreatin falls off adherent stem cell from culture bottle wall digestion, because there are many protein moleculars for cell surface, to avoid excessively digesting
The change that morphosis or function are caused to cell needs to add terminate liquid after pancreatin digestion a period of time is added in culture bottle
To terminate enzymic digestion effect.
In actual experiment operating process, conventionally used terminate liquid is the complete medium for being added to pancreatin inhibitor,
This method is easy to operate, and termination works well, and for most colleges and universities, Research Center, the laboratories such as hospital are used in one's power.But
Import complete medium is at high price, on a small quantity using still receiving under experiment condition, for mass propgation stem cell for clinic
When research etc., culture medium cost becomes inevitable problem.In addition, we are in practice, it has been found that use addition pancreatin
When the complete medium of inhibitor terminates enzymic digestion effect, because disposable digestion quantity is more, postdigestive stem cell is being operated
It will appear again adherent phenomenon in the process, this makes the stem cell of recycling have a certain number of reductions, causes to waste.
Summary of the invention
In order to solve the problems mentioned above in the background art, the present invention provides a kind of raising umbilical cord mesenchymal stem cells
The digestion method of the rate of recovery, comprising the following steps: be added and contain in the umbilical cord mesenchymal stem cells mixed liquor of Xiang Hanyou digestive pharmaceutical
The physiological saline of ALB terminates pancreatin digestion.
Preferably, the digestive pharmaceutical is the pancreatin PBS solution that quality volume fraction is 0.25%.
Preferably, the physiological saline containing ALB is the ALB physiological saline of mass volume ratio 5mg/ml-8mg/ml.
Preferably, the pH value of used PBS solution is 7.2-7.4.
Preferably, the mass fraction of used physiological saline is 0.9%.
The beneficial effects of the present invention are:
1) present invention improves the rate of recovery of umbilical cord mesenchymal stem cells after digestion.2) it is dry thin that present invention reduces umbilical cord mesenchymas
The toxigenic capacity of born of the same parents is conducive to umbilical cord mesenchymal stem cells mass propgation, to meet the scientific research etc. of bigger demand.
Detailed description of the invention
Fig. 1 is A group mescenchymal stem cell of the present invention sampling streaming testing result figure.
Fig. 2 is B group mescenchymal stem cell of the present invention sampling streaming testing result figure.
Fig. 3 is the amplification sample figure of each chart in Fig. 1 and Fig. 2.
Specific embodiment
The embodiment of the present invention is described in detail below, so that advantages and features of the invention can be easier to by ability
Field technique personnel understanding, so as to make a clearer definition of the protection scope of the present invention.
English abbreviation illustrates:
ALB: albumin；
PBS: phosphate buffered saline solution.
Embodiment
1, key instrument, reagent, consumptive material
Instrument: Thermo CO2 incubator, Thermo centrifuge, SD-WJ-2HD superclean bench, Accuri C6plus streaming
Cell instrument, JSY-SC-021H cell counter.
Reagent: GENVIEW PBS, Gibco pancreatin, Gibco penicillin/streptomycin, Helios serum, Gibco MEM α
Culture medium, CD antibody, heparin sodium, the ALB solution that mass fraction is 20%, mass fraction are 0.9% physiological saline, pH7.2 PBS.
Consumptive material: T175 Tissue Culture Flask, 10ml-50ml pipette, 50ml centrifuge tube, cell counting board.
2, method and step
(1) P2 for freezing laboratory recovers for umbilical cord mesenchymal stem cells, in 37 DEG C, 5%CO in T175 Tissue Culture Flask2Training
It supports to passage when merging about 80%-90% and spreads cultivation to P4 generation；
It (2) is 0.25% with quality volume fraction after being washed with pH7.2PBS when P4 is fused to about 80%-90% for cell growth
Pancreatin PBS solution digest and collect counting, be seeded to several T175 culture bottles, every bottle of inoculation according to 10^4/cm2 density
Cell total amount is (1.75 ± 0.13) × 10^6, culture bottle is divided into two groups of A, B at random, every group of 6 culture bottles；
(3) A, B group culture bottle are placed in 37 DEG C, 5%CO simultaneously2It is cultivated under the same terms；
(4) it when two groups of cell growth fusion about 80%-90% of A, B, is washed repeatedly 3 times, is thoroughly washed away not adherent with pH7.2PBS
Cell, impurity and culture medium；
(5) add the pancreatin PBS solution that the quality volume fraction of 4ml is 0.25% to each culture bottle of A, B group, culture bottle horizontal is existed
Workbench digests 1min, and gently culture bottle is patted in side, and cell is made to fall off from bottle wall；
(6) the MEM α complete medium that 6ml contains pancreatin inhibitor is added into A group culture bottle respectively, into B group culture bottle
The physiological saline that 10ml 5mg/ml containing ALB is added terminates pancreatin digestion；
(7) cell in each bottle of A, B group is collected respectively to 50ml centrifuge tube, 1600rpm is centrifuged 5min, removes supernatant；
(8) precipitating is resuspended with 15ml pH7.2 PBS, is mixed, and 1600rpm is centrifuged 5min, is removed supernatant, is repeated 1 times；
(9) precipitating is resuspended with 50ml pH7.2 PBS, mixes, samples this 100ul from each solencyte of A, B group respectively and do cell count
And flow cytometer detection.
3, result
Cell counts show that each culture bottle cell quantity average out to (6.69 ± 0.20) × 10^7 of A group is a, each culture of B group
Bottle cell quantity average out to (7.12 ± 0.12) × 10^7, significant (P the < 0.05) (table of two groups of culture cell quantity othernesses
1)；In terms of Cell viability, each culture bottle Cell viability average out to (91% ± 3%) of A group, each culture bottle cell quantity of B group is averaged
For (91% ± 2%), two groups of Cell viability no significant differences (P > 0.05) (table 2).
Table 1: cell counts statistics
Table 2: Cell viability result statistics
Sampling streaming testing result show, two groups of A, B each bottle cell surface marker CD29+, CD90+, CD105+, CD34-,
CD45- ratio all in 95% or more (Fig. 1, Fig. 2), illustrates all high expression mescenchymal stem cell surface marker of two groups of cells of A, B
CD29+, CD90+, CD105+ do not express or express less hemopoietic stem cell surface marker CD34-, CD45-, it was demonstrated that two groups of A, B
Cell is mescenchymal stem cell.
From experimental result as can be seen that application production process involved in largely prepare stem cell when, using the present invention compared with
Conventional method can obtain further amounts of stem cell, avoid it is again adherent after digestion caused by loss cell, and through the present invention
The stem cell of harvest, Cell viability and cell phenotype are unaffected, and cell state is good.
The foregoing description of the disclosed embodiments, only for can be realized professional and technical personnel in the field or use this
Invention.Various modifications to these embodiments will be readily apparent to those skilled in the art, institute herein
The General Principle of definition can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore,
The present invention will not be limited to the embodiments shown herein, and is to fit to special with principles disclosed herein and novelty
The consistent widest scope of point.
Claims (5)
1. a kind of digestion method for improving the umbilical cord mesenchymal stem cells rate of recovery, it is characterised in that: the following steps are included: to containing
The physiological saline containing ALB is added in the umbilical cord mesenchymal stem cells mixed liquor of digestive pharmaceutical and terminates pancreatin digestion.
2. a kind of digestion method for improving the umbilical cord mesenchymal stem cells rate of recovery according to claim 1, it is characterised in that:
The digestive pharmaceutical is the pancreatin PBS solution that quality volume fraction is 0.25%.
3. a kind of digestion method for improving the umbilical cord mesenchymal stem cells rate of recovery according to claim 1, it is characterised in that:
The physiological saline containing ALB is the ALB physiological saline of mass volume ratio 5mg/ml-8mg/ml.
4. a kind of digestion method for improving the umbilical cord mesenchymal stem cells rate of recovery according to claim 2, it is characterised in that:
The pH value of used PBS solution is 7.2-7.4.
5. a kind of digestion method for improving the umbilical cord mesenchymal stem cells rate of recovery according to claim 3, it is characterised in that:
The mass fraction of used physiological saline is 0.9%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103157099A (en) * | 2011-12-19 | 2013-06-19 | 吴宗贵 | Mixed enzyme digestive juice for fast digestion of vascular adventitia and preparation method thereof |
| CN104212761A (en) * | 2014-08-20 | 2014-12-17 | 北京瑞思德生物科技有限公司 | Kit for preparing placenta stem cells |
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2019
- 2019-02-27 CN CN201910145681.3A patent/CN109628395A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103157099A (en) * | 2011-12-19 | 2013-06-19 | 吴宗贵 | Mixed enzyme digestive juice for fast digestion of vascular adventitia and preparation method thereof |
| CN104212761A (en) * | 2014-08-20 | 2014-12-17 | 北京瑞思德生物科技有限公司 | Kit for preparing placenta stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 黄晓花: "公民逝世后器官捐献供肾肾移植围手术期间充质干细胞输注效果观察及护理", 《山西医药杂志》 * |
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Application publication date: 20190416 |