AU2021104393A4 - Method for sequential culture of human umbilical cord mesenchymal stem cells with triple mediums - Google Patents
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- 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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Abstract
A method for sequential culture of human umbilical cord mesenchymal stem
cells with triple mediums. The present invention provides a sequential culture method
using the triple mediums. The primary isolation culture method of umbilical cord
MSCs in this invention adopts a first medium mainly composed of DMEM with high
glucose, fetal bovine serum and antibiotics. Sterile coverslips are placed on the tissue
blocks to promote the adherence of the tissue blocks and the growth of mesenchymal
stem cells. When the cells are cultured with the first medium until the cell growth
state is stable, a second medium is used for subculture. The second medium is
composed of DMEM high glucose medium, fetal bovine serum, antibiotics and
mesenchymal stem cell growth additives. Then, the first medium is replaced again for
culture after two subcultures. The experiment proved that the MSCs isolated and
cultured by this method could be used in the related research fields of regenerative
medicine. The method in the present invention greatly reduces the cost of isolation
and cultivation of mesenchymal stem cells, with simple steps and high success rate,
which is suitable for large-scale cultivation of human umbilical cord mesenchymal
stem cells.
Description
[0001] The present invention relates to the technical field of stem cell culturing, and
particularly to a method for sequential culture of human umbilical cord mesenchymal
stem cells with triple mediums.
[0002] Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells derived
from mesoderm and ectoderm, with self-renewal and multidirectional differentiation
potential. MSCs are widely found in bone marrow, fat, placenta, menstrual blood,
umbilical cord blood and umbilical cord tissue, and have been deemed to be a
valuable source of cells in the field of gene therapy. MSCs from different sources
have similar morphology and surface markers, as well as similar biological properties,
but have different number of cells and different isolation methods. Human umbilical
cord MSCs are derived from the umbilical cord Wharton's jelly and perivascular
tissue. Human umbilical cord MSCs have broad tissue sources for easy acquisition,
which is convenient for collection and transportation. Human umbilical cord MSCs
have low immunogenicity and stable biological properties, without allogeneic
rejection. Furthermore, it is relatively simple to isolate and culture human umbilical
cord MSCs, and there is no ethical restriction, as well as no harm to donors.
Consequently, human umbilical cord MSC is gradually becoming an ideal choice and
one of the research hotspots in promising medical applications of stem cells.
[0003] The human umbilical cord is a jellied connective tissue having a length of 60
centimeters and a weight of 40 grams. Researchers are having trouble isolating
umbilical cord MSCs from umbilical cord tissue in a simple, economical and efficient
way. At present, the main methods for primary separation of human umbilical cord
MSCs are the enzyme digestion method and the tissue block adherent method.
Enzyme digestion method requires the use of a variety of enzymes, and some enzymes such as collagenase may involve animal sources, the price of which is expensive and the economic cost is high. In addition, the operation of this method is complicated and the digestion time is long, so it is difficult to control the digestion degree. Especially, the long-term digestion time is likely to damage cells, resulting in the death of a large number of cells and a low cell yield. The operation of the tissue block adherent method is simple and easy, with a low economic cost, but the crawling speed of cells is too slow, and the time of primary culture is too long, which is easy to cause cell aging and insufficient cell purity, thus affecting the clinical application of human umbilical cord MSCs.
[0004] The Chinese patent CN 109337867 B discloses an isolation method for
umbilical cord MSCs, which combines the tissue block adherent method and the
enzyme digestion method. This method can improve the isolation efficiency and
increase the number of mesenchymal stem cells, but the use of collagenase, selected
culture medium and growth factors lead to a high cost, which is not conducive to
large-scale production of mesenchymal stem cells.
[0005] The purpose of the present invention is to provide a method for sequential
culture of human umbilical cord mesenchymal stem cells with triple mediums. This
method not only improves the isolation efficiency and increases the number of
mesenchymal stem cells, but also greatly reduces the cost of isolation and culture of
human umbilical cord mesenchymal stem cells, which is suitable for large-scale
production of mesenchymal stem cells.
[0006] In order to accomplish the above purpose, the present invention adopts the
following technical solution:
[0007] A method for sequential culture of human umbilical cord mesenchymal stem
cells with triple mediums includes the following steps:
[0008] 1) separating Wharton's jelly;
[0009] 2) washing the Wharton's jelly tissue blocks with normal saline, cutting the
tissue blocks into the size of1-2mm3 , spreading the tissue blocks evenly on a culture plate pre-coated with gelatin at 37C for 2h, and then adding the first medium for culture, while placing sterile coverslips on the tissue blocks to promote the adherence of the tissue blocks and the growth of mesenchymal stem cells; the first medium is mainly composed of Dulbecco's Modified Eagle Medium (DMEM) with high glucose, fetal bovine serum and antibiotics, of which the volume fraction of fetal bovine serum is 7.5-15%;
[0010] 3) when the cells are cultured with the first medium until the cell growth state
is stable, then a second medium is used for subculture; the second medium is mainly
composed of DMEM with high glucose, fetal bovine serum, antibiotics and
mesenchymal stem cell growth additives, of which the volume fraction of fetal bovine
serum is 7.5-15%, and the volume fraction of mesenchymal stem cell growth
additives is 0.1-0.3%.
[0011] 4) cultivating the mesenchymal stem cells with the first medium again after
two subcultures.
[0012] Preferably, the separation of Wharton's jelly in step 1) includes the following
steps: obtaining a fetal umbilical cord tissue under aseptic conditions, washing the
fetal umbilical cord tissue with sterile saline, discarding the deformed parts of vessels
and both opening ends of the fetal umbilical cord tissue, tearing the umbilical cord,
removing two arteries, one vein and the outer membrane of the umbilical cord with
tissue forceps, and retaining an intima, the intima is the Wharton's jelly. Preferably,
the umbilical cord tissue is taken from a normal full-term fetus delivered by cesarean
section.
[0013] Preferably, the step of adding the first medium for culture instep 2) includes,
statically culturing for the first 3 days, adding 3-4ml of the first medium on the fourth
day, observing the growth situation, subsequently replacing the first medium every
2-3 days, removing the tissue blocks and replacing the first medium when most tissue
blocks generate crawl-out cells.
[0014] Preferably, the culture plate pre-coated with the gelatin in step 2) is coated
overnight at 4C.
[0015] Preferably, culturing cells with the first medium until the cell growth state of cells is stable in step 3) refers to reaching a 70-80% of cell fusion degree of the mesenchymal stem cells by using the first medium for the primary culture.
[0016] Preferably, the contents of the antibiotics in the first medium and the second
medium are 100-150U/mL of penicillin, 100-150 g/mL of streptomycin and
0.25-0.75[tg/mL of amphotericin B.
[0017] Preferably, the culture condition described in steps 2) to 4) is 37°C and 5%
CO 2 .
[0018] The present invention has the following advantages:
[0019] The present invention provides a method for sequential culture of human
umbilical cord mesenchymal stem cells with triple mediums. Unlike the traditional
tissue block adherent method, the primary isolation culture method of umbilical cord
MSCs in the present invention adopts a first medium consisting mainly of DMEM
with high glucose, fetal bovine serum, and antibiotics, and places sterile coverslips on
the tissue blocks to promote the adherence of the tissue blocks and the growth of
mesenchymal stem cells. The experiment showed that the growth rate of umbilical
cord tissue with sterile coverslips on it is significantly faster than that without sterile
coverslips in the process of primary isolation culture with the same medium, and the
cell density is significantly higher than that without sterile coverslips. The cell
crawl-out time and primary culture time are shorter than those of the tissue blocks
without sterile coverslips group (P < 0.05). In addition, in the process of subculture,
when the cells are cultured with the first medium until the cell growth state is stable, a
second medium (contains MSCs growth additive) is used for subculture twice, and
then the first medium is used again for subsequent subculture. There is no commercial
MSC-specific medium used throughout the process, and from the 4th generation until
the 1 5 th generation, a common medium is sufficient for cell proliferation and growth.
The raw material costs of 2"d generation MSCs and 6th generation MSCs are
calculated separately, where the cultivation cost of 2"d generation MSCs cost is about
1100 RMB, and that of 6th generation MSCs is about 300 RMB. The method in the
present invention greatly reduces the cost of isolation and cultivation of human
umbilical cord mesenchymal stem cells, with simple steps and high survival rate, which is suitable for large-scale production of mesenchymal stem cells. Moreover, the results of the proliferation ability detection and adipogenic differentiation test verify that the MSCs isolated and cultured by the method of the present invention can be fully used in the related research fields of regenerative medicine, which lays a firm foundation for the better application of umbilical cord mesenchymal stem cells.
[0020] FIG. 1 is a diagram showing the cell morphology of MSCs at the 5 th day of
primary culture (200 X );
[0021] FIG. 2 is a diagram showing the cell morphology of MSCs at the 1 4th day of
primary culture (200 X );
[0022] FIG. 3 is a diagram showing the cell morphology of MSCs at the 10 th day of
primary culture obtained from embodiment 1 and comparative example 1 (200 X);
[0023] In the FIG. 3, A shows tissue blocks without sterile coverslips; B shows
tissue blocks with sterile coverslips on the surface;
[0024] FIG. 4 is a diagram showing the cell morphology of MSCs at the 3rd day of
primary culture obtained from embodiment 1 and comparative example 2 (200x);
[0025] In the FIG. 4, A shows the 2"d generation cells cultivated by DMEM
high-glucose medium for 3 days; B showing the 2"d generation cells cultivated by
triple mediums for 3 days;
[0026] FIG. 5 is a diagram showing the growth curves of MSCs after primary
culture and subculture;
[0027] FIG. 6 is a diagram showing the flow cytometry results of MSCs obtained in
embodiment 1;
[0028] FIG. 7 is a diagram showing the adipogenic differentiation detection result of
MSCs obtained in embodiment 1 (200 X).
[0029] The present invention is further described in combination with the specific
embodiments below, but the scope of protection of the present invention is not limited to this. The instruments, equipment, and reagent materials used in the embodiment and experimental examples, unless specified, are all commercially available.
Embodiment 1
[0030] The embodiment provides a method for sequential culture of human
umbilical cord mesenchymal stem cells in umbilical cord tissue samples with triple
mediums. Collection of umbilical cord tissue: with the consent of the puerpera and
their families, the umbilical cord tissue of the healthy puerpera at 38-40 weeks of
cesarean section is collected in the Third Affiliated Hospital of Xinxiang Medical
University. The umbilical cord tissue is placed in a pre-filled 100ml D-Hanks
equilibrium salt solution under clean conditions in the operating room, and then, seal
and transport to the laboratory at 2°C-8°C. The specific steps are as follows:
[0031] 1. Separation of Wharton's jelly
[0032] An umbilical cord tissue of the normal full-term fetus delivered by cesarean
section is obtained under aseptic conditions. The umbilical cord tissue is cleaned
several times with sterile saline until the blood stains are washed, and the deformed
parts of vessels and both opening ends of the umbilical cord tissue are discarded. The
umbilical cord tissue is cut into small sections of 3cm-5cm. The umbilical cord is torn.
Two arteries, one vein and the outer membrane of the umbilical cord are removed
with tissue forceps, while the inner membrane, that is Wharton's jelly, is retained, and
then wash in normal saline 2-3 times.
[0033] 2. Cultivation of tissue blocks: the tissue blocks of Wharton's jelly are cut
into the size of 1-2mm3 and evenly spread in the culture plate pre-coated with gelatin
at 37C for 2h, and then the appropriate amount of the first medium is added into the
culture plate, and sterile coverslips are placed on the tissue blocks to promote the
adherence of tissue blocks and the growth of mesenchymal stem cells. After that, the
culture plate is placed in an incubator at 37C and 5% CO 2 .
[0034] The culture plate is statically cultured for the first 3 days.
[0035] 3-4ml of the first medium is added on the fourth day, and the growth is
observed. After that, the first medium is replaced every 2-3 days.
[0036] The first medium is mainly composed of DMEM with high glucose, fetal bovine serum and antibiotics, in which the volume of the fetal bovine serum accounted for 10%. In other embodiments, an optional range of the volume fraction of the fetal bovine serum in the first medium is 7 .5-15%, for example, 7 .5%, 12%, or %. The antibiotic contents are 100-150U/mL of penicillin, 100-150 g/mL of streptomycin, and 0.25-0.75 g/mL of amphotericin B.
[0037] 3. The tissue blocks are removed and the first medium is replaced when cells have crawled out from most of the issue blocks. When the cell density reaches %-80%, the second medium is used for subculture for two sequential generations.
[0038] The second medium is mainly composed of DMEM with high glucose, fetal bovine serum, antibiotics and mesenchymal stem cell growth additives. The volume fraction of the fetal bovine serum is 10%. In other embodiments, an optional range of the volume fraction of the fetal bovine serum in the second medium is 7.5-15%, for example, 7 .5%, 12%, or 15%. The volume fraction of the mesenchymal stem cell
growth additives is 0.2%. In other embodiments, the volume fraction of the fetal bovine serum in the second medium is within the optional range of 0.1-0.3%, e.g., 0.1%, 0.3%. The antibiotic contents are 100-150U/mL of penicillin, 100-150 g/mL of
streptomycin, and 0.25-0.75 g/mL of amphotericin B.
[0039] 4. The cells are placed back to the first medium for subsequent cultivation after 4 th generation. Comparative example 1
[0040] In this example, umbilical cord tissue samples are also used for mesenchymal stem cell culture, and the collection of the umbilical cord tissue is shown in embodiment 1. In the process of primary isolation and cultivation in this example, the same medium as embodiment 1 is used, but no sterile coverslips are placed on the umbilical cord tissue. The specific steps are as follows:
[0041] 1. Separation of Wharton's jelly (See embodiment 1)
[0042] 2. Cultivation of tissue blocks: the tissue blocks of Wharton's jelly are cut into the size of1-2mm3 and evenly spread in the culture plate pre-coated with gelatin at 37C for 2h, and then the appropriate amount of the first medium is added into the culture plate. After that, the culture plate is placed in an incubator at 37C and 5%
CO 2 .
[0043] The culture plate is statically cultured for the first 3 days.
[0044] 3-4ml of the first medium is added on the fourth day, and the growth is
observed. After that, the first medium is replaced every 2-3 days.
[0045] The first medium is mainly composed of DMEM with high glucose, fetal
bovine serum and antibiotics, in which the volume fraction of the fetal bovine serum
is 10%. The antibiotic contents are 100-150U/mL of penicillin, 100-150 g/mL of
streptomycin, and 0.25-0.75 g/mL of amphotericin B.
[0046] 3. The tissue blocks are removed and the first medium is replaced when cells
have crawled out from most of the issue blocks. When the cell density reaches
%-80%, the second medium is used for subculture for two sequential generations.
[0047] 4. The cells are placed back to the first medium for subsequent cultivation
after 4 th generation.
[0048] The second medium is mainly composed of DMEM with high glucose, fetal
bovine serum, antibiotics and mesenchymal stem cell growth additives. The volume
fraction of the fetal bovine serum is 10%. The volume fraction of the mesenchymal
stem cell growth additives is 0.2%. The antibiotic contents are 100-150U/mL of
penicillin, 100-150 g/mL of streptomycin, and 0.25-0.75 g/mL of amphotericin B.
Comparative example 2
[0049] In this example, normal DMEM with high glucose is used for the cultivation
of mesenchymal stem cells in umbilical cord tissue samples. Umbilical cord tissue
collection is shown in embodiment 1, and the specific steps are as follows:
[0050] 1. Separation of Wharton's jelly (See embodiment 1)
[0051] 2. Cultivation of tissue blocks: the tissue blocks of Wharton's jelly are cut
into the size of1-2mm3 and evenly spread in the culture plate pre-coated with gelatin
at 37C for 2h and then the DMEM with high glucose is added into the culture plate.
After that, the culture plate is placed in an incubator at 37C and 5% CO 2 .
[0052] The culture plate is statically cultured for the first 3 days.
[0053] 3-4ml of the DMEM with high glucose is added on the fourth day, and the
growth is observed. After that, the DMEM with high glucose is replaced every 2-3 days.
[0054] The DMEM high glucose medium is mainly composed of DMEM, fetal
bovine serum and antibiotics, in which the volume fraction of the fetal bovine serum
is 10%. The antibiotic contents are 100-150U/mL of penicillin, 100-150tg/mL of
streptomycin, and 0.25-0.75 g/mL of amphotericin B.
[0055] 3. The tissue blocks are removed and the DMEM with high glucose is
replaced when cells have crawled out from most of the issue blocks. When the cell
density reaches 70%-80%, subsequent subculture is continued on the DMEM with
high glucose.
Experimental example
[0056] In this example, human umbilical cord mesenchymal stem cells in the
umbilical cord tissue samples are cultured by using the method for sequential culture
of human umbilical cord mesenchymal stem cells with the triple mediums provided in
embodiment 1, and the methods provided in comparative example 1 and comparative
example 2 are used as comparisons such that a series of identification of cultured
umbilical cord mesenchymal stem cells are conducted by observing the cell growth
status, drawing in vitro growth curve of umbilical cord mesenchymal stem cells, using
the flow cytometry to detect cell surface markers, and detecting the adipogenic
differentiation ability.
[0057] (I) Morphological characteristics and cell yield of MSCs
[0058] The morphological characteristics of the primary culture cells are observed
under an inverted phase contrast microscope, the cultivation results of the method in
embodiment 1 are shown in FIG. 1 and FIG. 2 respectively. As shown in FIG. 1,
MSCs have crawled out of the tissue blocks on the 5th day of primary culture. As
shown in FIG. 2, the degree of cell fusion could reach 70%-80% on the 1 4th day of
primary culture.
[0059] The difference between comparative example 1 and embodiment 1 is that
there are no sterile coverslips on the tissue blocks of comparative example 1. The cell
morphology observation and statistical analysis of MSCs at the 10 th day of primary
culture are performed, and the results are shown in FIG. 3 and Table. 1. As can be seen from the figure, the cell density of tissue blocks with sterile coverslips covered
(as shown in FIG. 3B) is significantly higher than that of tissue blocks without sterile
coverslips (as shown in FIG. 3A). In addition, the crawl-out time and primary culture
time of the tissue blocks with sterile coverslips (embodiment 1) are shorter than those
of the tissue blocks without sterile coverslips (comparative example 1); those
differences are with statistical significance (P< 0. 05) (Table. 1).
[0060] Table. 1 Comparison of MSCs culture effects between the two groups
Tissue Blocks With/Without Cell Crawl-out time (h) Primary Culture Time
Sterile Coverslips (d)
Without (Comparative example) 139.5±6.2 21.5±2.8
With (Embodimentl) 96.2±4.6* 15.2±2.3*
[0061] Note: Compared with the group without sterile coverslips, *P < 0. 05
[0062] The difference between comparative example 2 and embodiment 1 is that
comparative example 2 is cultured in the common DMEM with high glucose. The cell
morphology of MSCs at the 3 rd day of subculture is observed, and the cell viability is
calculated with the help of a trypan blue solution. The calculation formula is as
follows: cell viability = number of living cells / (number of dead cells + number of
living cells) X 100, and the result is as shown in FIG. 4 and Table. 2. The cell density
and cell viability of the 2"d generation cells cultured for 3 days by the method using
the triple mediums are significantly higher than those that cultured for 3 days by the
common method using the DMEM with high glucose, which means the cell yield of
the method using the triple mediums is significantly better than that of the common
method using the DMEM high-glucose.
[0063] Table. 2 Comparison of culture rate of MSCs between the two groups
Group Cell Density Cell Viability
Comparative example 2 23.2% 80.8%
Embodiment 1 36.3%* 92.5%*
[0064] Note: Compared with the comparative example, *P < 0. 05
[0065] (II) Growth curve of MSCs
[0066] The 2"d, 6 th and 1 2th generations of MSCs are taken and prepared into
single-cell suspension separately, and the cell concentration is adjusted to 5x104 /mL.
The single-cell suspension is inoculated into a 24-well plate at the rate of 200L/well,
and cultured in an incubator at 37°C, 5% CO 2 and saturated humidity. From the 2d
day, two wells are randomly selected for counting, and the average value is taken as
the cell number of the day. The MSCs are observed and counted continuously for 12
days, and the in vitro growth curve of MSCs is plotted as shown in FIG. 5. The
growth curve shows that there is no significant change among the proliferation rate
and the growth cycle of the 2d, 6th and 1 2th generations of MSCs. Specifically, the
first to third days are the incubation period of cell growth, and from the fourth day on,
the cells enter into the logarithmic growth phase and start proliferation in quantity. It
could be observed under an inverted microscope that the cell protrusions continually
extend to the surrounding areas, the cell density increases gradually, and the cells are
connected to each other. The cell proliferation reaches the peak on the 10th day, then
slows down and enters the plateau phase.
[0067] (III) Phenotypic identification of MSCs
[0068] Taking the 6th generations of MSCs and digestion with 0.25% trypsin, then
the 6th generation of MSCs are centrifuged at 1000rpm for 10min, and the supernatant
is discarded. The MSCs are washed and precipitated with phosphate buffered saline
(PBS) 3 times, and 1x106 cells are counted by re-suspending. 20pL of fluid monoclonal antibody is added separately, including CD45-FITC and CD44-FITC. The
MSCs are placed at room temperature for 30min in the dark room and then
centrifuged at 1000 rpm for 5min. The MSCs are resuspended by adding 4 0 0 pL of
PBS after removing the unbound antibodies, and then detects by flow cytometry. The
specific operation is carried out in accordance with the operation rules of flow
cytometry.
[0069] The phenotype identification results of flow cytometry are shown in FIG. 6,
indicating that the stem cell marker CD44 can be expressed stably and uniformly in
the MSCs isolated and cultured by the method provided by the present invention,
while the marker antigen CD45 of hematopoietic precursor cells is hardly expressed.
[0070] (IV) Differentiation potential test of MSCs
[0071] The 6 th generation of MSCs are taken to be prepared into single-cell
suspension, and then is inoculated in a 6-well plate. The 6-well plate is placed in an
incubator at 37°C, 5% CO2 and saturated humidity for culture. When 80% of the cells
are fused, the cell fluid is replaced with the medium A for adipogenic induction and
differentiation of human umbilical cord mesenchymal stem cells. After 3 days of
induction, the medium A is discarded and replaced with the medium B for adipogenic
induction differentiation of human umbilical cord mesenchymal stem cells. After 24
hours, the medium B is replaced with the medium A. The alternate cycle is executed 3
times about 12 days, and then the cells are cultured with the medium B for 3 days
until the lipid droplets become large and round. After the current adipogenesis
induction and differentiation, 4% neutral formaldehyde solution is used for fixation.
The 6-well plate is placed under an inverted microscope to observe the staining effect
of lipid droplets after staining with 0.3% oil red 0 staining solution. The results after
differentiation culture and oil red 0 staining are shown in FIG. 7. FIG. 7 shows that
lipid droplets are formed in all MSCs, indicating that MSCs isolated and cultured by
this method still has multidirectional differentiation potential after multiple rounds of
subcultures.
[0072] Based on the above experimental results, the cells isolated and cultured using
the method for human umbilical cord mesenchymal stem cells cultivation of the
present invention are confirmed to be human umbilical cord mesenchymal stem cells,
and the method for culturing human umbilical cord mesenchymal stem cells of the
present invention is sample. There is no commercial MSC-specific medium used
throughout the process, and from the 4th generation to the 15th generation, a common
medium is sufficient for cell proliferation and growth, which greatly reduces the cost
of isolation and cultivation of mesenchymal stem cells. Thus, this method is suitable
for cultivation of large-scale production of mesenchymal stem cells for treatment of
related diseases.
Claims (8)
1) separating Wharton's jelly;
2) washing Wharton's jelly tissue blocks with normal saline, cutting the tissue
blocks into a size of 1-2mm3 , spreading the tissue blocks evenly on a culture plate
pre-coated with gelatin at 37C for 2h, adding a first medium for culture; during the
culture process, placing sterile coverslips on the tissue blocks to promote an
adherence of the tissue blocks and a growth of mesenchymal stem cells; the first
medium is mainly composed of Dulbecco's Modified Eagle Medium (DMEM) with
high glucose, fetal bovine serum and antibiotics, in which a volume fraction of the
fetal bovine serum is 7.5-15%;
3) when the cells are cultured with the first medium until the cell growth state is
stable, placing the tissue blocks in a second medium for subculture; the second
medium is mainly composed of DMEM with high glucose, the fetal bovine serum, the
antibiotics and mesenchymal stem cell growth additives, in which the volume fraction
of the fetal bovine serum is 7.5-15%, and a volume fraction of the mesenchymal stem
cell growth additives is 0.1-0.3%;
4) cultivating the mesenchymal stem cell with the first medium again after two
subcultures.
2. The method according to claim 1, characterized in that the step of separating
the Wharton's jelly in step 1) includes the following steps: obtaining a fetal umbilical
cord tissue under aseptic conditions, washing the fetal umbilical cord tissue with
sterile saline, discarding deformed parts of vessels and both opening ends of the fetal
umbilical cord tissue, tearing the umbilical cord, removing two arteries, one vein and
outer membrane of the umbilical cord by tissue forceps, and retaining an intima, the
intima is Wharton's jelly.
3. The method according to claim 2, characterized in that the umbilical cord
tissue is taken from a normal full-term fetus delivered by cesarean section.
4. The method according to claim 1, characterized in that the step of adding the
first medium for culture in step 2) includes: statically culturing for the first 3 days,
adding 3-4ml of the first medium on the fourth day, observing the growth of the
mesenchymal stem cells, subsequently replacing the first medium every 2-3 days,
removing the tissue blocks and replacing the first medium when the mesenchymal
stem cells crawled out of most tissue blocks.
5. The method according to claim 1, characterized in that the culture plate
pre-coated with gelatin in step 2) is coated overnight at 4°C.
6. The method according to claim 1, characterized in that culturing cells with the
first medium until the cell growth state is stable in step 3) refers to reaching a 70-80%
of cell fusion degree of the mesenchymal stem cells by primary culture.
7. The method according to claim 1, characterized in that the contents of the
antibiotics of the first medium and the second medium are 100-150 U/mL of penicillin,
100-150 g/mL of streptomycin and 0.25-0.75[tg/mL of amphotericin B.
8. The method according to claim 1, characterized in that a culture condition in
steps 2) to 4) is 37°C and 5% Co 2 .
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| CN202110579979.2A CN113201490A (en) | 2021-05-26 | 2021-05-26 | Method for sequentially culturing human umbilical cord mesenchymal stem cells by triple culture medium |
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| WO2008073331A2 (en) * | 2006-12-07 | 2008-06-19 | Teva Pharmaceutical Industries Ltd. | Method of generation and expansion of tissue-progenitor cells and mature tissue cells from intact bone marrow or intact umbilical cord tissue |
| CN102559590B (en) * | 2012-01-16 | 2013-11-06 | 遵义医学院附属医院 | Method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media |
| CN104673743A (en) * | 2015-02-13 | 2015-06-03 | 中国人民解放军第二军医大学 | Tissue block culture method for obtaining primary cell from animal tissue |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
| CN110731969A (en) * | 2019-10-27 | 2020-01-31 | 北京中广天一生物科技有限公司 | Preparation of mesenchymal stem cells and application of mesenchymal stem cells in preparation of pain medicines |
| CN111235100B (en) * | 2020-02-21 | 2022-04-29 | 新乡医学院 | Culture method of human umbilical cord blood mesenchymal stem cells |
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