CN112195150A - Method for improving primary acquisition of umbilical cord mesenchymal stem cells - Google Patents
Method for improving primary acquisition of umbilical cord mesenchymal stem cells Download PDFInfo
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- CN112195150A CN112195150A CN202011141060.7A CN202011141060A CN112195150A CN 112195150 A CN112195150 A CN 112195150A CN 202011141060 A CN202011141060 A CN 202011141060A CN 112195150 A CN112195150 A CN 112195150A
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- 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
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- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The embodiment of the invention discloses a method for improving primary acquisition of umbilical cord mesenchymal stem cells, belonging to a method for separating and culturing stem cells, comprising the steps of plate laying, fluid infusion, fluid replacement and the like, wherein the temperature is 37 ℃, and the CO content is 5 percent2Is cultured in a carbon dioxide incubator by using Gibco TryPLETMThe pancreatin substitute is digested, human serum albumin is adopted to stop the digestion, the cell climbing amount and the cell harvesting amount are high, the clone formation rate is high, and the form is good.
Description
Technical Field
The invention relates to a method for separating and culturing stem cells, in particular to a method for improving primary acquisition of umbilical cord mesenchymal stem cells.
Background
In recent years, cell drug research and development become hot spots of drug research and development, and stem cells from different sources have come into the market in foreign countries. The umbilical cord mesenchymal stem cell acquisition and amplification technology has a mature research system and application, but stable large-scale preparation is still a difficult point to be solved in commercial production, especially in the primary cell acquisition stage.
At present, two methods for obtaining primary cells are mainly used, namely an enzymolysis method and a bottle climbing method. The enzymolysis method has more operation steps, relates to the back-and-forth penetration among various instruments, and has large production control risk; the related biological enzyme is difficult to evaluate the cell damage, the introduced bioactive substance residue is difficult to detect, and the bioreactor is mostly derived from microorganisms such as escherichia coli, so that the safety risk is increased. In the pharmaceutical industry, the control of raw materials for production makes this process not widely applicable.
Most culture containers adopted by bottle climbing production are culture dishes or culture bottles with air-permeable covers, the culture dishes are simple and convenient to operate, but the culture dishes are large in opening and cannot be sealed, so that microbial pollution is easily caused in the operation, transfer and culture processes; the culture bottle can better reduce the pollution risk, but the operation difficulty is correspondingly increased because the opening is smaller. The climbing rate and climbing-out amount of the bottle climbing method are difficult to stabilize, the main reason is that the cells are distributed in the Huatong glue tissue unevenly, and the method cannot help to improve the adherent proliferation of the cells, so that the primary acquisition amount of the bottle climbing method is generally smaller than that of an enzymolysis method and is not high in stability.
Some bio-instrument manufacturers have recently introduced instruments that can automate the handling of the umbilical cord. The instruments generally have fewer functions, only act on a certain stage of primary operation, such as the operation of the obtained Wharton jelly, the flux is smaller, the single umbilical cord treatment capacity is lower, the single treatment lasts for several hours, the price is higher, the single instrument exceeds 10 ten thousand yuan, and some instruments still need to be combined with biological enzymes to play a better role, so the instruments are not suitable for being applied to large-scale preparation.
Disclosure of Invention
The invention aims to solve the technical problems of small primary acquisition amount and large pollution risk of stem cells in the prior art, and provides a method for improving the primary acquisition of umbilical cord mesenchymal stem cells.
The technical scheme provided by the invention is as follows:
a method for improving primary acquisition of umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
(1) plate paving: inoculating the tissue blocks into a cell culture bottle, and adding primary culture solution to uniformly attach the tissue blocks to the surface of the cell culture bottle;
(2) liquid supplementing: after 3-7 days of culture, taking out a cell culture bottle, supplementing 2-10 mL of primary culture solution preheated by 37 ℃ water bath, and continuing to culture for 3-7 days;
(3) liquid changing: sucking out culture waste liquid, adding 10mL of primary culture liquid into each bottle, continuing culturing, and carrying out subculture when the cell climbing condition of not less than 10 tissue blocks and at least 1 visual field magnified by 40x of each tissue block reaches the following condition: the number of the tissue blocks successfully crawled out in each cell bottle is more than 15, and the cell crawling amount of each tissue block can occupy a 40 multiplied by visual field.
Preferably, the amount of the inoculated tissue block in the step (1) is 0.25-0.75 ml, and the amount of the inoculated culture solution is 2-12 ml.
Preferably, the culture environment is 37 ℃ and 5% CO2The carbon dioxide incubator of (1).
Preferably, the primary cell harvesting method comprises the steps of:
a, cleaning: the tissue block is pulled down, the tissue block and culture waste liquid are poured out, the cell surface is rinsed for 2 times by using cleaning liquid, 10mL of the cleaning liquid is added each time, and the surface on which cells grow cannot be directly rinsed when liquid is added;
b, digestion: adding 2mL of digestive juice, observing under a microscope, and adding 2mL of stop solution to stop digestion when cracks appear between cells and shrinkage begins;
c, centrifugal counting: centrifuging the cell suspension at 270 Xg for 5min, discarding the supernatant, and resuspending the cell pellet to obtain primary cells (P)0)。
Preferably, the digestive juice is Gibco TryPLETMPancreatin substitute, wherein the stop solution is human blood albumin.
Preferably, before the plating, the umbilical cord treatment is carried out, and the umbilical cord treatment comprises the following steps:
a, cleaning: squeezing the umbilical cord out of residual blood in a culture dish of 150mm by using a pair of tweezers, cleaning for 3 times or more by using a cleaning solution, wherein each time is not less than 20mL, and cutting the umbilical cord into small sections of 1-3 cm by using scissors until no obvious blood color exists;
b, removing: cutting off the vein by using scissors, and gently scraping the vein wall by using forceps; finding out and removing two arterial blood vessels; the Wharton jelly was removed with forceps and placed in a 50mL centrifuge tube containing 20mL of primary culture medium.
c, cutting into pieces: cleaning with cleaning solution at a speed of 1200rpm for 5min for 3 times (no less than 20mL each time), discarding the supernatant, and cutting with ophthalmic scissors to pieces of about 1-2mm3The tissue mass of (1).
By adopting the technical scheme, the technical effects are as follows:
the method is simple, has low pollution risk, greatly increases the primary acquisition amount, and has good form and high cloning efficiency.
Drawings
FIG. 1 is a graph comparing the results of different primary harvesting methods, wherein a is tissue mass adherence method, b is collagenase digestion method, and c is composite collagenase digestion method;
FIG. 2 is a comparison of the cell passage effect of different primary acquisition methods, where A is tissue mass adherence, B is collagenase digestion, and C is complex collagenase digestion.
Detailed Description
In order to further understand the technical solution of the present invention, the following description is given with reference to the examples.
Examples
Firstly, an umbilical cord is treated, wherein the umbilical cord treatment comprises the following steps:
a, cleaning: squeezing the umbilical cord out of residual blood in a culture dish of 150mm by using a pair of tweezers, cleaning for 3 times or more by using a cleaning solution, wherein each time is not less than 20mL, and cutting the umbilical cord into small sections of 1-3 cm by using scissors until no obvious blood color exists;
b, removing: cutting off the vein by using scissors, and gently scraping the vein wall by using forceps; finding out and removing two arterial blood vessels; the Wharton jelly was removed with forceps and placed in a 50mL centrifuge tube containing 20mL of primary culture medium.
c, cutting into pieces: cleaning with cleaning solution at a speed of 1200rpm for 5min for 3 times (no less than 20mL each time), discarding the supernatant, and cutting with ophthalmic scissors to pieces of about 1-2mm3The tissue mass of (1).
Secondly, culturing the tissue block, comprising the following steps:
(1) plate paving: inoculating 0.5ml tissue block into cell culture bottle, adding 10ml primary culture solution to make tissue block uniformly adhere to cell culture bottle surface, and culturing at 37 deg.C and 5% CO2Culturing in a carbon dioxide incubator;
(2) liquid supplementing: after 7 days of culture, taking out a cell culture bottle, supplementing 8mL of primary culture solution preheated by 37 ℃ water bath, and continuing to culture for 7 days;
(3) liquid changing: sucking out culture waste liquid, adding 10mL of primary culture liquid into each bottle, continuing culturing, and carrying out subculture when the cell climbing condition of not less than 10 tissue blocks and at least 1 visual field magnified by 40x of each tissue block reaches the following condition: the number of the tissue blocks successfully crawled out in each cell bottle is more than 15, and the cell crawling amount of each tissue block can occupy a 40 multiplied by visual field.
Finally, primary cells are obtained, the obtaining method comprising the steps of:
a, cleaning: the tissue block is pulled down, the tissue block and culture waste liquid are poured out, the cell surface is rinsed for 2 times by using cleaning liquid, 10mL of the cleaning liquid is added each time, and the surface on which cells grow cannot be directly rinsed when liquid is added;
b, digestion: adding 2mL of Gibco TryPLETM pancreatin substitute digestive juice, observing under a microscope, and adding 2mL of human serum albumin stop solution to stop digestion when cracks appear among cells and shrinkage begins;
c, centrifugal counting: centrifuging the cell suspension at 270 Xg for 5min, discarding the supernatant, and resuspending the cell pellet to obtain primary cells (P)0)。
Comparative example
The primary treatment methods of umbilical cord reported in literature and commonly used at present are mainly divided into 3 types: tissue block adherent culture, collagenase digestion and composite collagenase digestion [ Yi Fu Hua, Yang Xiao Feng, Huang Sheng Man, et al, comparison of the isolation and preparation method of human umbilical cord mesenchymal stem cells 3 [ J ]. clinical examination J, 2010, 028(006):413-414 ]. The tissue block adherent culture method is a method for naturally climbing out tissue blocks. The collagenase digestion method is a method in which cells of an umbilical cord tissue mass are digested with enzymes to digest the cells of the tissue mass into small cells or cell masses, and the cells are continuously expanded to obtain primary cells. The compound collagenase digestion method is a method for increasing enzyme types for digestion on the basis of a collagenase digestion method, thereby achieving better acquisition efficiency and quality.
According to the comparative experiment results in the literature, the trypsinization method under the same conditions can not successfully separate and culture the MSCs. The reason may be that the umbilical cord tissue has complicated composition and content, is difficult to digest by enzymes, or has poor controllability in the digestion process, resulting in poor separation stability. [ sinus comet, Guo wenjun, Yuli, et al, research on human umbilical cord mesenchymal stem cell isolation culture method [ J ]. China J. histochemistry and cytochemistry, 10.3870/zgzzhx.2010.02.016] enzyme has an action mechanism of decomposing protein and colloid, exposing MSCs therein so as to be easily collected, but the action of the enzyme has no specificity and directionality, is limited by various reasons in the operation process, is very easy to damage membrane-bound protein and other structures on the cell surface, generates unpredictable damage to cells, and finally causes poor cell state and even death. The collagenase digestion method and the compound collagenase digestion method have poor controllability, the risk is difficult to observe, the cell quality is seriously influenced after the risk occurs, the type and the degree of the influenced quality cannot be predicted, and the requirements of the production process on the process stability are not met. Digestion of the enzymes used would also present a safety risk. Pancreatin drugs inquired from the state drug administration are all oral dosage forms, and injection dosage forms are not inquired, so that pancreatin or other enzymes are avoided as much as possible in the selection of the production process except the condition of necessary use. The collagenase digestion method and the compound collagenase digestion method have poor applicability in the production of cell medicines.
Aiming at the reported results of the literature, the experiments in the literature [ Yi Fu Hua, Yang Xiao Feng, Huang Sheng Man, et al, comparison of 3 separation and preparation methods of human umbilical cord mesenchymal stem cells [ J ]. J. J.clinical examination, 2010, 028(006):413-414.] are repeated, the cells are subjected to clone formation rate detection, and data are collected and evaluated.
Results of the experiment
As can be seen from the above table, from the view point of climbing out the primary tissue mass, as can be seen from fig. 1, the tissue mass adherence method in this embodiment is best, and the form is most normal (spindle-shaped, spiral growth) with the largest climbing-out amount; collagenase digestion only allows sporadic cell crawl out and survive, and reproductive activity seems to be damaged and not amplified any more; the complex collagenase digestion method has cell creeping out and growth, but the growth condition is general. The technical scheme of the invention overcomes the defects of a collagenase digestion method in the prior art, has higher creep amount and good cell morphology.
From the perspective of cell passage effect: the obtained primary cells were further cultured, morphologically observed, and the cell harvest amount of each culture vessel (T75 cell culture flask) was calculated and the colony formation rate was measured. As can be seen from FIG. 2, the tissue mass adherence method of the present embodiment has the highest cell yield, better clone formation rate and better cell morphology. The embodiment of the invention overcomes the defects of the collagenase digestion method in the prior art, not only obtains cells with good shapes, but also obviously improves the cell yield and the clone formation rate.
Claims (6)
1. A method for improving primary acquisition of umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
(1) plate paving: inoculating the tissue blocks into a cell culture bottle, and adding primary culture solution to uniformly attach the tissue blocks to the surface of the cell culture bottle;
(2) liquid supplementing: after 3-7 days of culture, taking out a cell culture bottle, supplementing 2-10 mL of primary culture solution preheated by 37 ℃ water bath, and continuing to culture for 3-7 days;
(3) liquid changing: sucking out culture waste liquid, adding 10mL of primary culture liquid into each bottle, continuing culturing, and carrying out subculture when the cell climbing condition of not less than 10 tissue blocks and at least 1 visual field magnified by 40x of each tissue block reaches the following condition: the number of the tissue blocks successfully crawled out in each cell bottle is more than 15, and the cell crawling amount of each tissue block can occupy a 40 multiplied by visual field.
2. The method for improving primary harvest of umbilical cord mesenchymal stem cells according to claim 1, wherein the amount of the tissue mass inoculated in step (1) is 0.25-0.75 ml, and the amount of the inoculated culture solution is 2-12 ml.
3. The method of improving primary harvest of umbilical cord mesenchymal stem cells according to claim 1, wherein the culture environment is 37 ℃ and 5% CO2The carbon dioxide incubator of (1).
4. The method of improving primary acquisition of umbilical cord mesenchymal stem cells according to claim 1, wherein the primary cell acquisition method comprises the steps of:
a, cleaning: the tissue block is pulled down, the tissue block and culture waste liquid are poured out, the cell surface is rinsed for 2 times by using cleaning liquid, 10mL of the cleaning liquid is added each time, and the surface on which cells grow cannot be directly rinsed when liquid is added;
b, digestion: adding 2mL of digestive juice, observing under a microscope, and adding 2mL of stop solution to stop digestion when cracks appear between cells and shrinkage begins;
c, centrifugal counting: centrifuging the cell suspension at 270 Xg for 5min, discarding the supernatant, and resuspending the cell pellet to obtain primary cells (P)0)。
5. The method of improving primary acquisition of umbilical cord mesenchymal stem cells according to claim 4, wherein the digestive fluid is Gibco TryPLETMPancreatin substitute, wherein the stop solution is human blood albumin.
6. The method of improving primary acquisition of umbilical cord mesenchymal stem cells according to claim 1, wherein prior to plating, an umbilical cord treatment is performed, the umbilical cord treatment comprising the steps of:
a, cleaning: squeezing the umbilical cord out of residual blood in a culture dish of 150mm by using a pair of tweezers, cleaning for 3 times or more by using a cleaning solution, wherein each time is not less than 20mL, and cutting the umbilical cord into small sections of 1-3 cm by using scissors until no obvious blood color exists;
b, removing: cutting off the vein by using scissors, and gently scraping the vein wall by using forceps; finding out and removing two arterial blood vessels; the Wharton jelly was removed with forceps and placed in a 50mL centrifuge tube containing 20mL of primary culture medium.
c, cutting into pieces: cleaning with cleaning solution for 3 times without washing each timeCentrifuging at 1200rpm for 5min under 20mL, discarding supernatant, and cutting into pieces of about 1-2mm3The tissue mass of (1).
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102660503A (en) * | 2012-05-22 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cells from umbilical cord |
| US20180362923A1 (en) * | 2015-12-11 | 2018-12-20 | Lei Guo | Method for separating and extracting huc-msc from wharton's jelly tissue of umbilical cord |
| CN109652368A (en) * | 2019-01-31 | 2019-04-19 | 和携科技(北京)有限公司 | The method of primary mescenchymal stem cell is obtained from umbilical cord tissue |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102660503A (en) * | 2012-05-22 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cells from umbilical cord |
| US20180362923A1 (en) * | 2015-12-11 | 2018-12-20 | Lei Guo | Method for separating and extracting huc-msc from wharton's jelly tissue of umbilical cord |
| CN109652368A (en) * | 2019-01-31 | 2019-04-19 | 和携科技(北京)有限公司 | The method of primary mescenchymal stem cell is obtained from umbilical cord tissue |
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