CN115094025A - Preparation method of endometrial stromal stem cells - Google Patents
Preparation method of endometrial stromal stem cells Download PDFInfo
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Abstract
The invention provides a preparation method of endometrial stromal cell, which relates to the technical field of stem cell preparation, and comprises the steps of menstrual blood collection, cell separation, cell culture, passage, inoculation, cell cryopreservation, preservation and the like.
Description
Technical Field
The invention relates to the technical field of stem cell preparation, in particular to a preparation method of endometrial stromal stem cells.
Background
The cell storage means that human cells are stored in vitro for a long time by using a deep low temperature cryopreservation method, and the function and activity of the cryopreserved cells are not influenced. Through cell storage, the self or variant young and healthy cells can be stored, and can be used in anti-aging or disease treatment, so that the body aging speed is delayed, the disease can be effectively treated, and further, the life health is ensured in advance.
For a long time, cell storage has been invasive, such as spinal cord aspiration, fat aspiration, wisdom tooth aspiration, and the like. The mesenchymal stem cells are obtained from the spinal cord tissue, which is an invasive process, causes certain pain to donors, and has limited number of obtained stem cells and long doubling time. The placenta tissue or umbilical cord blood stem cells can only be collected at birth and are in short supply, and the clinical application of the mesenchymal stem cells is seriously influenced by the limiting factors.
Therefore, due to the limitation and restriction of various conditions such as stem cell extraction time, an acquisition mode, an acquisition scene (most of the stem cells need to be acquired in medical institutions) and the like, the price of the Chinese cell storage industry is high (2-3 ten thousand yuan), audience groups are greatly limited, the number of cells capable of being extracted is also limited, and if the actual content of each cord blood hematopoietic stem cell is less than 0.5%, the actual requirement of future clinical treatment is difficult to meet.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a preparation method of endometrial stromal stem cells, which can be used for large-scale collection and application, can realize non-invasive collection of a full-flow sample, has small damage to the sample and strong proliferation capacity of stem cells, and the prepared stem cells have high purity.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides a preparation method of endometrial stromal stem cells, which comprises the following steps:
(a) collecting menstrual blood: collecting menstrual blood by using a menstrual blood collecting kit, wherein the menstrual blood collecting kit comprises an incubator, a collecting tube, endometrium protective solution, a menstrual cup and an ice box; collecting menstrual blood with a menstrual cup, pouring all menstrual blood into a collecting tube, adding endometrium protective solution, screwing down a collecting tube cover, storing at 2-8 ℃, putting the collecting tube with menstrual blood and an ice box into an incubator, and transporting to a laboratory within 24-48 hours;
wherein the endometrium protective solution is based on an endometrium matrix stem cell culture medium and contains 5-10 mM NAC, 50-100 mu g/mL streptomycin, 1-5 mu M staurosporine and 1000-2000U/mL LIF;
the endometrium matrix stem cell culture medium is based on 95% DMEM and 5% serum and contains 5-10 ng/mL of insulin, 1-5 mu M of staurosporine, 1-10 mu M A-83-01, 50-100U/mL of penicillin and 50-100 mu g/mL of streptomycin;
(b) cell separation: centrifuging a collection tube filled with menstrual blood, adding endometrial stem cell separation fluid, blowing and beating into tissue suspension, standing, centrifuging, discarding supernatant, adding red blood cell lysate with 5-6 times volume, cracking for 3-5 minutes, centrifuging, removing supernatant, adding PBS, blowing and beating into cell suspension, and centrifuging;
wherein the endometrial stroma stem cell separation solution is based on DMEM and contains 1.4-2.0 mu mol/L adenosine triphosphate, 1-5 mu M staurosporine, 50-100U/mL penicillin and 50-100 mu g/mL streptomycin;
(c) cell culture, passage and inoculation: centrifuging, then removing the supernatant, adding the endometrium matrix stem cell culture medium, inoculating cell precipitates into a culture bottle, placing the culture bottle in a constant-temperature constant-humidity incubator containing 5-6% of carbon dioxide at 37-38 ℃, changing the culture solution every 2-3 days, and carrying out cell passage when the polymerization degree of the cells reaches 80-90%;
discarding an old endometrial stromal cell culture medium, adding PBS for washing, then adding 0.25-0.3% of pancreatin-EDTA for digestion for 1-2 min, adding 2-3 times of the endometrial stromal cell culture medium to stop digestion, transferring the endometrial stromal cell culture medium into a centrifugal tube, centrifuging, discarding supernatant, adding PBS for blowing and beating to form cell suspension, centrifuging, discarding supernatant, adding the endometrial stromal cell culture medium for heavy suspension and precipitation, and blowing and beating to form single cell suspension;
according to the number of subculture, uniformly blowing and beating the cells, then respectively inoculating the cells into new culture bottles, uniformly shaking the cells in a cross manner, and then putting the cells into a constant-temperature constant-humidity incubator containing 5-6% of carbon dioxide at 37-38 ℃ for culture until the cell fusion degree (polymerization degree) reaches 80-90%;
(d) freezing and storing cells: discarding the old endometrial stroma stem cell culture medium, adding PBS for washing, then adding 0.25-0.3% of pancreatin-EDTA for digestion for 1-2 min, adding 2-3 times of the endometrial stroma stem cell culture medium to stop digestion, transferring the endometrial stroma stem cell culture medium into a centrifugal tube, centrifuging, discarding the supernatant, adding PBS for blowing and beating into cell suspension, centrifuging, discarding the supernatant, and resuspending the sediment by using frozen stock solution; sub-packaging the cell suspension into a freezing tube;
(e) freezing and storing in a warehouse: and (5) preserving the frozen tube containing the cells.
(a) Collecting menstrual blood
The menses collecting suit consists of an incubator, a sample collecting tube, endometrium protective solution, a menstruation cup, an ice box and the like, and can keep the menses sample at a refrigerating temperature (2-8 ℃) all the time within 24-48 hours.
The endometrium protective solution is based on an endometrium matrix stem cell culture medium and contains 5-10 mM NAC, 50-100 mu g/mL streptomycin, 1-5 mu M staurosporine and 1000-2000U/mL LIF.
Wherein the endometrium matrix stem cell culture medium is based on 85-95% DMEM and 5-15% serum, and contains 5-10 ng/mL insulin, 1-5 mu M staurosporine, 1-10 mu M A-83-01, 50-100U/mL penicillin and 50-100 mu g/mL streptomycin.
Endometrium protective liquid
NAC is N-acetyl-L-cysteine in an amount of, for example, 5, 6, 7, 8, 9, 10mM typical but not limiting of endometrial stromal stem cell culture media.
The streptomycin content is typically, but not limited to, 50, 60, 70, 80, 90, 100. mu.g/mL of the endometrial stromal stem cell culture medium.
The content of the staurosporine in the culture medium of the endometrial stromal stem cells is typically, but not limited to, 1, 2, 3, 4, 5. mu.M.
LIF is a leukemia inhibitory factor, and is typically present in an amount, but not limited to, 1000, 1500, 1800, 2000U/mL of endometrial stromal stem cell culture medium.
The endometrium protecting liquid has the functions of maintaining the bioactivity of the intrauterine membrane tissue cell inside the menstrual blood without loss, inhibiting the growth of the flora inside the menstrual blood of the vulval tract and eliminating mycoplasma pollution. The main role of LIF: through a JAK/STAT signal channel, the dryness of the endometrial stem cells is maintained, and the undifferentiated state is kept; major roles of staurosporine: division and proliferation of the endometrial stem cells are mainly promoted by means of the staurosporine, and the stem cells cannot replicate by themselves without the staurosporine.
Further, in the step (a), 10-50 mL of the endometrial protection solution is added to every 10-50 mL of menstrual blood, and the volume ratio of 1: 1.
(b) cell separation
Adenosine triphosphate content is typical but not limiting of DMEM, for example 1.4, 1.5, 1.6, 1.7, 1.8, 2.0 μmol/L.
The content of staurosporine is typically, but not limited to, 1, 2, 3, 4, 5. mu.M in DMEM.
Penicilin is Penicillin, typically but not limited to DMEM, for example 50, 60, 70, 80, 90, 100U/mL.
Streptomyces is Streptomycin, which is typically present in amounts such as, but not limited to, 50, 60, 70, 80, 90, 100. mu.g/mL of DMEM.
The endometrium stroma stem cell separating medium has the functions of decomposing and dissociating stem cell components in endometrium tissues shed in menstrual blood, collecting the stem cell components, and recovering the cells with the cell viability rate of over 90 percent by adopting the separating medium.
(c) Cell proliferation
Endometrium matrix stem cell culture medium
Insulin levels are typically, but not limited to, 5, 6, 7, 8, 9, 10 ng/mL in DMEM + serum, for example.
The content of staurosporine is typically, but not limited to, 1, 2, 3, 4, 5. mu.M in DMEM + serum.
A-83-01 is a small molecule compound belonging to the ALK signaling pathway inhibitor, and the content of the culture medium in DMEM + serum is typically but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 μ M.
Penicilin content is typically, but not limited to, 50, 60, 70, 80, 90, 100U/mL of DMEM + serum, for example.
Streptomyces content is typical of DMEM + serum but is for example, but not limited to, 50, 60, 70, 80, 90, 100. mu.g/mL.
The endometrium contains a plurality of cell types such as vascular endothelial cells, epithelial cells, interstitial cells, stromal stem cells and the like, and the specially developed endometrium stromal stem cell culture medium can be used for specifically screening the stromal stem cell type with a repairing effect in the process of cell enrichment culture, while the rest cell types can not proliferate and survive in the culture medium. The purity of the finally harvested endometrial stroma stem cells can reach 99.6 percent through detection of a flow cytometer.
(d) Cell cryopreservation
In a preferred embodiment, the components of the cryopreservation solution comprise 90-95% of the above endometrial stromal stem cell culture medium and 5-10% of dimethyl sulfoxide.
The matrix stem cell freezing protective solution has the function of preventing ice crystals in cells from forming in the process of cooling and freezing storage so as to avoid cell damage and death.
(e) Frozen and stored in a warehouse
In a preferred embodiment, step (e) comprises: placing a freezing tube containing cells into a refrigerator at 4-5 ℃ for precooling for 20-30 min; then putting the freezing tube containing the cells into a precooled isopropanol cooling box, transferring the box into an ultra-low temperature refrigerator at minus 80 ℃, and standing for 10 hours; the next day, the cryopreservation tube containing the cells is transferred into a gas-phase liquid nitrogen tank for long-term storage.
The cells are frozen, namely a matrix stem cell programmed cooling method, the temperature of the endometrial matrix stem cells is reduced from 4 ℃ to-80 ℃ by using an isopropanol cooling box for only 9.8 hours (a programmed cooling control system reduces the temperature at a constant speed of one degree every 7 minutes). The freezing storage and recovery efficiency of the stem cells can reach more than 95 percent.
The invention has at least the following beneficial effects:
the invention obtains stem cells from the exfoliated tissue of menstrual blood source, has convenient material acquisition, does not need the help of professionals, and has multiple steps of sample receiving, tissue inoculation, amplification culture, cryopreservation and warehousing and the like, successfully establishes a complete high-efficiency technical system, can realize the noninvasive collection of a full-flow sample, has small damage to the sample and strong proliferation capacity of the stem cells (the stem cells are amplified by one time every 24 hours on average) by improving the endometrium protective solution, the endometrium stroma stem cell separating solution, the endometrium stroma stem cell culture medium and the cryopreservation method, and has the purity of the prepared stem cells reaching 99.60 percent, which is the highest standard of the purity of the cells in the same industry. The stem cells from menstrual blood sources become the autologous adult stem cells which have the most advantages and potential and can be collected and applied in a large scale.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the actual time required for detecting one-fold expansion of endometrial stromal stem cells in a cell doubling assay in accordance with example 1 of the present invention.
FIG. 2 is a graph showing the actual time required for detecting one-fold expansion of endometrial stromal stem cells in the comparative example 6 cell doubling assay of the present invention.
FIG. 3 is a graph of the flow cytometry results of the purity of endometrial stromal stem cells obtained from example 1 of the present invention, wherein a is the stem cell population, b is CD90-CD 105-blank control, c is CD90-CD34-CD105-CN73-, d is CD73+ CD105+, e is CD73+ CD90+, f is CD90+ CD34-, g is isotype control, and h is HLA-DR-.
FIG. 4 is a graph showing the results of the differentiation potency test of endometrial stromal stem cells according to example 1 of the present invention, wherein the left side shows the results of osteogenic differentiation (100X), the middle part shows the results of chondrogenic differentiation (100X), and the right side shows the results of adipogenic differentiation (100X).
FIG. 5 shows the result of the proliferation potency test of endometrial stromal stem cells according to example 1 of the present invention.
FIG. 6 is a graph of the raw data of the flow cytometry detection of endometrial stromal stem cells of the comparative example 5 of the present invention, wherein a is the stem cell population, b is CD90-CD 105-blank control, c is CD90-CD34-CD105-CN73-, d is CD90+ CD105+, e is CD73+ CD105+, f is CD105+ CD34-, g is isotype control, and h is HLA-DR-.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The invention is further illustrated by the following examples. The materials in the examples are prepared according to known methods or are directly commercially available, unless otherwise specified.
Example 1
The composition and content of the reagents used in example 1 were as follows:
the endometrium matrix stem cell culture medium is based on 95% DMEM and 5% serum and contains 5 ng/mL of insulin, 2 mu M of staurosporine, 5 mu M A-83-01, 100U/mL of penicillin and 100 mu g/mL of streptomycin;
the endometrium protective solution is based on endometrium matrix stem cell culture medium and contains 10mM NAC, 100 mu g/mL streptomycin, 2 mu M staurosporine and 1000U/mL LIF;
the endometrial stroma stem cell separation solution is based on DMEM and contains 1.4 mu mol/L adenosine triphosphate, 2 mu M staurosporine, 100U/mL penicilin and 100 mu g/mL streptomycin;
the frozen stock solution comprises 90% of the endometrial stromal stem cell culture medium and 10% of dimethyl sulfoxide.
1. The menses collecting set using method comprises the following steps:
(1) freezing and temporary storage of endometrial protection fluid: after receiving the collection set, the ice box is put into a refrigerator for freezing (-18 ℃) for more than 24 hours; the endometrium protective solution is put into a refrigerator for refrigeration (4 ℃) for preservation. The rest materials and the box body are properly preserved at room temperature for standby.
(2) Waiting for menstrual cycle, selecting acquisition time: collecting menstrual blood in the time period with large menstrual flow.
(3) Collecting menstrual blood: collecting menstrual blood with a menstrual cup, and pouring all menstrual blood into a collecting tube.
(4) Adding a protective liquid: pouring the endometrium protective solution into the collecting tube according to the actual menstrual blood volume and the equal volume ratio, and screwing the cover of the collecting tube.
(5) Temporary storage: the collecting pipe is put into a refrigerator (4 ℃) for temporary storage, so that the vertical and stable collecting pipe is ensured.
(6) Filling in an acquisition information card: filling personal data and menstrual blood collection information in detail.
(7) Boxing: and placing the frozen ice bag into a sample collection suit according to the original position, and placing a collection tube filled with menstrual blood, a completely filled collection information card and a physical examination report into the sample collection suit.
(8) Handover: and after the collection is finished, the cold-chain logistics entryway receives the sample and mails the sample to a cell preparation engineering center for preparing and storing the stem cells.
2. Endometrial stromal stem cell storage:
(1) the sample collection tube containing menstrual blood was removed from the collection set and centrifuged at 1500rpm for 5 min.
(2) After centrifugation, 10ml of endometrial stem cell separation fluid is added and blown into tissue suspension, and after standing for 5-10 minutes, centrifugation is carried out at 1500rpm for 5 min.
(3) After centrifugation, the supernatant is discarded, 5 times volume of erythrocyte lysate is added, lysis is carried out for 3 minutes, centrifugation is carried out for 5 minutes at 1500rpm, the supernatant is removed, PBS is added, cell suspension is blown and beaten, and centrifugation is carried out for 5 minutes at 1500 rpm.
[ cell viability assay ]
Total number of live cells/(total number of live cells + total number of dead cells) × 100%. test method: uniformly mixing the centrifuged resuspended single cell suspension with trypan blue solution in a ratio of 9:1, taking 10 mul of the suspension to be dropped on a cell counting plate, counting live cells and dead cells, and then counting the cell survival rate: living cell rate (%) =
And (3) testing results: the cell viability rate was (93.79. + -. 0.77)%. (as shown in Table 1)
(4) Discarding the supernatant, adding an endometrium matrix stem cell culture medium, inoculating the cell precipitate into a culture bottle, and placing the culture bottle in a constant-temperature constant-humidity incubator at 37 ℃ and 5% carbon dioxide. Changing the solution every 2-3 days, and carrying out cell passage when the degree of polymerization of the cells reaches 80-90%.
(5) Cell passage: discarding the old cell culture medium, adding PBS to wash twice, then adding 0.25% pancreatin-EDTA to digest for 1-2 min, adding 2 times of the volume of the culture medium to stop digestion, transferring the cell culture medium into a centrifugal tube by using a pipette, centrifuging the cell culture medium for 5min at 1500rpm, discarding the supernatant, adding PBS to blow and beat the cell culture medium into a cell suspension, centrifuging the cell culture medium for 5min at 1500rpm, discarding the supernatant, adding a proper amount of the culture medium to resuspend and precipitate, and blowing and beating the cell suspension to form a single cell suspension.
[ cell doubling time test ]
The test method comprises the following steps:
after cell subculture, counting cells at each time point of 0h,2h,4h,6h,8h,10h,12h,16h,18h,20h,22h,24 h,36h and 48h, and statistically analyzing the time required for cell amplification to be doubled.
And (3) testing results:
as shown in fig. 1, the time required for the endometrial stromal stem cells to multiply once on average is 22 hours.
(6) Cell inoculation: according to the number of subculture, cells are evenly blown and then respectively inoculated into new culture bottles, and the new culture bottles are evenly shaken in a cross manner and then placed into a constant-temperature constant-humidity incubator with 37 ℃ and 5% carbon dioxide for culture until the cell fusion degree reaches 80-90%.
[ purity test of endometrial stromal stem cells ]
The test method comprises the following steps:
the purity of the cells can be detected by flow cytometry by staining and labeling the stem cells with stem cell-specific labeling molecules, such as CD73, CD90, CD105, etc., and then loading the cells on a machine, analyzing the cells by flow cytometry while expressing the stem cell-specific labeling moleculesThe actual cell proportion of the molecule, and thus the purity value of the stem cell. The method comprises the following specific steps: the stem cells in culture were dissociated and digested into a cell suspension (1X 10) 6 and/mL), washing for 2 times by using PBS, adding stem cell specific marker molecules CD73-APC (5 mu L), CD90-FITC (5 mu L), CD105-Percp (5 mu L) and negative control CD34-PE (5 mu L), incubating for 30min at room temperature in a dark place, washing for 2 times by using PBS, adding 300 mu L of PBS, re-suspending, and detecting by using a flow cytometer.
And (3) testing results:
the cell purity of the endometrial stromal stem cells is 99.6 percent through flow cytometry detection.
The raw data for streaming analysis is shown in figure 3.
[ test of differentiation Capacity of endometrial stromal Stem cells ]
The test method comprises the following steps:
osteogenic differentiation of stem cells: taking P6 as a substitute for endometrial stromal stem cells, and replacing the endometrial stromal stem cells with 1-2 multiplied by 10 6 And inoculating the cells in a 6-well culture plate, and when the cell fusion degree reaches 50% -60% fusion, replacing an endometrium stromal cell culture medium with an osteogenesis induction culture medium, namely adding 0.1-1 mu M dexamethasone, 10-20 mM beta-sodium glycerophosphate and 50-100 mu g/ml ascorbic acid into a DMEM/F12 culture medium containing 10% -15% serum. The liquid was changed every 3 days. After 3 weeks of osteogenic induction, 4% paraformaldehyde was fixed and osteoblasts were identified by von kusa staining.
Chondrogenic differentiation of stem cells: replacing the endometrial stromal stem cells with P6 cells at a ratio of 2-3 × 10 5 The culture medium is inoculated in a 15ml centrifuge tube, centrifuged at 1500rpm for 10 min, supernatant is removed, and a finished cartilage induction culture medium is added, namely, 0.1-1 mu M dexamethasone, 50-100 mu g/ml ascorbic acid, 125-150 mu g/ml bovine serum albumin, 6.25-7.5 mu g/ml transferrin, 1-5 mM sodium pyruvate and 5.35-6.5 mu g/ml linoleic acid are added into a DMEM/F12 culture medium. The liquid was changed every 3 days. After 3 weeks of chondrogenic induction, the chondrocyte pellets were frozen and sectioned after fixation with 4% paraformaldehyde, and cells were identified by staining with alpha blue.
Adipogenic differentiation of stem cells: taking P6 as a substitute for endometrial stromal stem cells, and replacing the cells with 1-2 × 10 6 Inoculating the cells in 6-well culture plate, and inoculating the cells when the cell fusion degree reaches 50% -60% fusionThe method comprises the steps of replacing an endometrium substrate stem cell culture medium with an adipogenesis induction culture medium, namely adding 1-3 mu M, IBMXO 5-10 mM of dexamethasone and 100-200 mu M of indomethacin into a DMEM/F12 culture medium containing 10% -15% serum. The liquid was changed every 3 days. And 3 weeks after adipogenic induction, using oil red O for staining identification.
And (3) testing results:
as shown in fig. 4, endometrial stromal stem cells have the biological properties of mesenchymal stem cells and can be directed to differentiation into bone, cartilage, fat, and the like.
(7) Freezing and storing cells: when the cell polymerization degree reaches 85-90%, discarding the old cell culture medium, adding PBS to wash twice, then adding 0.25% pancreatin-EDTA to digest for 1-2 min, adding 2 times of volume of culture medium to stop digestion, transferring to a centrifuge tube by a pipette, centrifuging for 5min at 1500rpm, discarding supernatant, adding PBS to blow and beat into cell suspension, centrifuging for 5min at 1500rpm, discarding supernatant, and resuspending the centrifuged cell precipitate with frozen stock solution. Freezing the cell suspension according to each tube 10 6 The specification standard of 1ml is subpackaged in a freezing tube. And sticking the frozen cell coding label on the subpackaged frozen tube.
(8) Freezing and storing in a warehouse: firstly, placing a freezing tube containing cells into a refrigerator at 4 ℃ for precooling for 20 min; then placing the freezing tube containing the cells into an isopropanol cooling box precooled in advance, transferring the freezing tube into an ultra-low temperature refrigerator at minus 80 ℃ and placing the freezing tube for 10 hours; the next day, the cryopreservation tube containing the cells is transferred into a gas-phase liquid nitrogen tank for long-term storage.
[ cell proliferation potency assay ]
The test method comprises the following steps:
and (3) comparing and analyzing the proliferation capacity of the endometrial stromal stem cells after the cryopreservation solution (cryopreservation solution) is stored and the endometrial stromal stem cells after the cryopreservation solution is not stored, and counting the cells at each time point of 0h,2h,6h,10h,16h,24h and 48h respectively, so as to analyze the cell proliferation capacity in the in-vitro culture process. As shown in fig. 5, it was found through experiments that the endometrial stromal stem cells preserved with the cryoprotectant had a stronger proliferation potency than the endometrial stem cells preserved with the non-cryoprotectant, and the difference was significant.
(9) Cell recovery: clamping the frozen cells in the liquid nitrogen by using forceps, putting into a 37-DEG C constant-temperature water bath kettle, and quickly dissolving the cells completely within 1 min; transferring the dissolved cell suspension into a 15ml centrifugal tube, simultaneously adding 5-10 ml DMEM/F12 culture medium, centrifuging for 5min at 1500rpm, discarding the supernatant, adding PBS to blow and beat into cell suspension, centrifuging for 5min at 1500rpm, discarding the supernatant, re-suspending the centrifuged cell sediment by using endometrium matrix stem cell culture medium, and blowing and beating to form single cell suspension. And according to the number of subcultures, uniformly blowing and beating the cells, respectively inoculating the cells into new culture bottles, uniformly shaking the cells, and culturing the cells in a constant-temperature constant-humidity incubator containing 37 ℃ and 5% of carbon dioxide until the cell fusion degree reaches 80-90%, thus carrying out subculture on the cells.
Example 2
This example differs from example 1 in that the endometrial protection fluid was replaced with: based on the endometrial stromal stem cell culture medium, 10mM NAC, 100. mu.g/mL streptomycin, 5. mu.M staurosporine and 1500U/mL LIF were contained.
[ cell viability assay ]
The test method was the same as in example 1;
and (3) testing results: the cell viability rate was (94.36. + -. 0.98)%. (as shown in Table 1)
Example 3
This example differs from example 1 in that the endometrial stem cell separation fluid was replaced with: DMEM-based, containing 2.0. mu. mol/L adenosine triphosphate, 5. mu.M staurosporine, 100U/mL penicillin and 100. mu.g/mL streptomycin.
[ cell viability assay ]
The test method was the same as in example 1;
and (3) testing results: the cell survival rate is (93.16 +/-1.64)%. (as shown in Table 1)
Comparative example 1
This example differs from example 1 in that the endometrial protection fluid is replaced by: based on the culture medium of endometrial stromal stem cells, the traditional Chinese medicine composition contains 10mM NAC and 100 mu g/mL streptomycin.
[ cell viability assay ]
The test method was the same as in example 1;
and (3) testing results: the cell survival rate is (57.56 +/-2.18)%. (as shown in Table 1)
Comparative example 2
This example differs from example 1 in that the endometrial protection fluid is replaced by: based on the culture medium of endometrial stromal stem cells, 10mM NAC, 100. mu.g/mL streptomycin and 1000U/mL LIF are contained.
[ cell viability assay ]
The test method was the same as in example 1;
and (3) testing results: the cell viability rate was (72.06. + -. 1.96)%. (as shown in Table 1)
Comparative example 3
This example differs from example 1 in that the endometrial stem cell separation fluid was replaced with: DMEM-based, containing 100U/mL penicillin and 100. mu.g/mL streptomycin.
[ cell viability assay ]
The test method was the same as in example 1;
and (3) testing results: the cell viability rate was (62.16. + -. 1.24)%. (as shown in Table 1)
Comparative example 4
This example differs from example 1 in that the endometrial stem cell separation fluid was replaced with: DMEM-based, containing 0.14. mu. mol/L adenosine triphosphate, 0.5. mu.M staurosporine, 100U/mL penicillin and 100. mu.g/mL streptomycin.
[ cell viability test ]
The test method was the same as in example 1;
and (3) testing results: the cell survival rate is (73.73 +/-1.18)%. (as shown in Table 1)
Comparative example 5
This example differs from example 1 in that the endometrial stromal stem cell culture medium is replaced with: based on 95% DMEM and 5% serum, 100U/mL penicillin and 100. mu.g/mL streptomycin were included.
[ cell purity test ]
The test method was the same as in example 1;
and (3) testing results:
the cell purity of the endometrial stromal stem cells is 60.18% as detected by flow cytometry.
The raw data for streaming analysis is shown in fig. 6.
Comparative example 6
This example differs from example 1 in that the endometrial stromal stem cell culture medium is replaced with: based on 95% DMEM and 5% serum, 10 ng/mL insulin, 100U/mL penicillin and 100. mu.g/mL streptomycin were included.
[ cell doubling time test ]
The test method was the same as in example 1;
and (3) testing results:
as shown in fig. 2, the time required for endometrial stromal stem cells to multiply once on average is 36 hours.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
1. A method for preparing endometrial stromal stem cells, comprising the steps of:
(a) collecting menstrual blood: collecting menstrual blood by using a menstrual blood collecting kit, wherein the menstrual blood collecting kit comprises an incubator, a collecting tube, endometrium protective solution, a menstrual cup and an ice box; collecting menstrual blood with a menstrual cup, pouring all menstrual blood into a collecting tube, adding endometrium protective solution, screwing down a collecting tube cover, storing at 2-8 ℃, putting the collecting tube with menstrual blood and an ice box into an incubator together, and transporting to a laboratory within 24-48 hours;
wherein the endometrium protective solution is based on an endometrium matrix stem cell culture medium and contains 5-10 mM NAC, 50-100 mu g/mL streptomycin, 1-5 mu M staurosporine and 1000-2000U/mL LIF;
the endometrium matrix stem cell culture medium is based on 85-95% DMEM and 5-15% serum and contains 5-10 ng/mL insulin, 1-5 mu M staurosporine, 1-10 mu M A-83-01, 50-100U/mL penicillin and 50-100 mu g/mL streptomycin;
(b) cell separation: centrifuging a collection tube filled with menstrual blood, adding endometrial stem cell separation fluid to blow and beat into tissue suspension, standing, centrifuging, discarding supernatant, adding erythrocyte lysate with 5-6 times volume of volume, cracking for 3-5 minutes, centrifuging, removing supernatant, adding PBS to blow and beat into cell suspension, and centrifuging;
wherein the endometrial stroma stem cell separation solution is based on DMEM and contains 1.4-2.0 mu mol/L adenosine triphosphate, 1-5 mu M staurosporine, 50-100U/mL penicillin and 50-100 mu g/mL streptomycin;
(c) cell culture, passage and inoculation: centrifuging, then removing the supernatant, adding the endometrium matrix stem cell culture medium, inoculating cell precipitates into a culture bottle, placing the culture bottle in a constant-temperature constant-humidity incubator with 37-38 ℃ and 5-6% carbon dioxide, changing the culture solution every 2-3 days, and carrying out cell passage when the degree of polymerization of the cells reaches 80-90%;
discarding an old endometrial stromal cell culture medium, adding PBS for washing, then adding 0.25-0.3% of pancreatin-EDTA for digestion for 1-2 min, adding 2-3 times of the endometrial stromal cell culture medium to stop digestion, transferring the endometrial stromal cell culture medium into a centrifugal tube, centrifuging, discarding supernatant, adding PBS for blowing and beating to form cell suspension, centrifuging, discarding supernatant, adding the endometrial stromal cell culture medium for heavy suspension and precipitation, and blowing and beating to form single cell suspension;
according to the number of subculture, uniformly blowing and beating the cells, respectively inoculating the cells into new culture bottles, uniformly shaking the cells, and culturing the cells in a constant-temperature constant-humidity incubator containing 5-6% of carbon dioxide at 37-38 ℃ until the cell fusion degree reaches 80-90%;
(d) freezing and storing cells: discarding the old endometrial stroma stem cell culture medium, adding PBS for washing, then adding 0.25-0.3% of pancreatin-EDTA for digestion for 1-2 min, adding 2-3 times of the endometrial stroma stem cell culture medium to stop digestion, transferring the endometrial stroma stem cell culture medium into a centrifugal tube, centrifuging, discarding the supernatant, adding PBS for blowing and beating into cell suspension, centrifuging, discarding the supernatant, and resuspending the sediment by using frozen stock solution; sub-packaging the cell suspension into a freezing tube;
(e) freezing and storing in a warehouse: and (5) preserving the frozen tube containing the cells.
2. The method for preparing according to claim 1, wherein the endometrium protective solution is based on an endometrial stromal stem cell culture medium, and comprises 10mM NAC, 100 μ g/mL streptomycin, 2 μ M staurosporine, and 1000U/mL LIF;
the endometrium matrix stem cell culture medium is based on 95% DMEM and 5% serum and contains 5 ng/mL insulin, 2 mu M staurosporine, 5 mu M A-83-01, 100U/mL penicillin and 100 mu g/mL streptomycin;
the endometrial stroma stem cell separating medium is based on DMEM and contains 1.4 mu mol/L adenosine triphosphate, 2 mu M staurosporine, 100U/mL penicillin and 100 mu g/mL streptomycin.
3. The method according to claim 1, wherein the components of the cryopreservation solution comprise 90 to 95% of the endometrial stromal stem cell culture medium and 5 to 10% of dimethyl sulfoxide.
4. The method of claim 3, wherein the components of the cryopreservation solution comprise 90% of the endometrial stromal stem cell culture medium and 10% dimethyl sulfoxide.
5. The method of claim 1, wherein step (e) comprises: placing a freezing tube containing cells into a refrigerator at 4-5 ℃ for precooling for 20-30 min; then putting the freezing tube containing the cells into a precooled isopropanol cooling box, transferring the box into an ultralow temperature refrigerator at minus 70 to minus 80 ℃, and standing for 10 to 15 hours; the next day, the cryopreservation tube containing the cells is transferred into a gas-phase liquid nitrogen tank for long-term storage.
6. The method according to any one of claims 1 to 5, wherein in step (a), 10 to 50mL of the endometrium protective solution is added per 10 to 50mL of menstrual blood.
7. The method according to claim 6, wherein in the step (a), the ratio of the volume of menstrual blood to the volume of the endometrial protection solution is 1: 1.
8. the method according to claim 6, wherein in the step (b), the ratio of the added volume of the endometrial stem cell separation fluid to the total volume of menstrual blood and the endometrial protection fluid is 1: 1.
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