CN106399247A - Purpose of umbilical cord mesenchymal stem cell exosome - Google Patents
Purpose of umbilical cord mesenchymal stem cell exosome Download PDFInfo
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- CN106399247A CN106399247A CN201610882018.8A CN201610882018A CN106399247A CN 106399247 A CN106399247 A CN 106399247A CN 201610882018 A CN201610882018 A CN 201610882018A CN 106399247 A CN106399247 A CN 106399247A
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- stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
Abstract
The invention relates to the field of stem cell culture, and particularly relates to a purpose of umbilical cord mesenchymal stem cell exosome. Shown by experiments, with respect to culture experiments adopting a complete medium, by adopting co-culture of the umbilical cord mesenchymal stem cell exosome and peripheral blood stem cells, the amount of cells acquired by culture can be increased by about 1.5 times, and a clone formation amount is also increased by about 1.5 times. Through statistical analysis, compared with a culture effect without the umbilical cord mesenchymal stem cell exosome, the experiment effect with addition of the umbilical cord mesenchymal stem cell exosome has an extremely remarkable difference (p is smaller than 0.001).
Description
Technical field
The present invention relates to stem cell culture field, the more particularly, to purposes of umbilical cord mesenchymal stem cells excretion body.
Background technology
Procedure for peripheral blood stem cells is the candidate stem cell being present in peripheral blood.Goldman in 1979 etc. is one group of acceleration
Gather frozen PBC when phase or the transplanting first visit of CML-BC patients with chronic myelocytic leukemia, so that patient is come back to slowly
The property phase, start the clinical practice of Transplantation of Peripheral Haemopoietic Stem Cells (PBSCT).
Under normal circumstances, the content of the ancestral cells in peripheral blood accounts for the 0.01%～0.1% of mononuclearcell and is equivalent to
The 1%～10% of marrow, CD34 in peripheral blood MNC after mobilization+Cell number and CFU number are obviously higher than marrow.Hitherto it is found that
Polyanion compound (as dextran sulfate), glucocorticoid, bacterial endotoxin, antineoplastic, tetrahydrofolic acid and
Hemopoieticgrowth factor etc. all can mobilize marrow and the hematopoietic stem/progenitor at other position to enter peripheral blood effectively.Thus have can
The enough stem cells required for stem cell transplantation directly can be collected from blood.But cell mobilization can be to candidate stem cell
Produce and damage, there is larger side effect.
If being capable of procedure for peripheral blood stem cells amount reproduction in vitro, can provide more to stem cell transplantation
Select, but at present, it is unsatisfactory that candidate stem cell expands numerous effect in vitro.
Content of the invention
In view of this, the technical problem to be solved in the present invention is to provide the purposes of umbilical cord mesenchymal stem cells excretion body,
Experiment shows, umbilical cord mesenchymal stem cells excretion body can improve the clonality of peripheral blood hematopoietic stem cells, promote outward
All blood hemopoietic stem cell proliferations and enhancing peripheral blood hematopoietic stem cells vigor.
The invention provides umbilical cord mesenchymal stem cells excretion body is in improving procedure for peripheral blood stem cells clonality
Application.
Present invention also offers application in promoting procedure for peripheral blood stem cells propagation for the umbilical cord mesenchymal stem cells excretion body.
Present invention also offers umbilical cord mesenchymal stem cells excretion body answering in strengthening procedure for peripheral blood stem cells vigor
With.
Excretion body (exosome is small vesica microvesicles again) is by endosome (endosomal
Compartment) discharge.The excretion body of umbilical cord mesenchymal stem cells (mesenchrymal stem cells, MSCs) passes through life
Thing synthesis and endocytosis participate in the accurately film transportation of complicated regulation and control.Existing research is proved the excretion of MSCs secretion
Body can reduce myocardial damage scope, protection acute tubular damages, it is important to promote the aspects such as nerve regneration, minimizing injury of lungs to have
Effect.Experiments indicate that, for the culture experiment using complete medium, using umbilical cord mesenchymal stem cells outside
Secrete body to co-culture with procedure for peripheral blood stem cells, the cell quantity of culture gained can be improved about 1.5 times, Clone formation quantity
Improve about 1.5 times.Through statistical analysis, compared with the culture effect being not added with umbilical cord mesenchymal stem cells excretion body, add navel
Experiment effect with mescenchymal stem cell excretion body has pole significant difference (p<0.001).
Present invention also offers a kind of culture medium of procedure for peripheral blood stem cells, it contains umbilical cord mesenchymal stem cells excretion
Body.
Specifically, the culture medium that the present invention provides includes：Volume fraction be 30%～35% umbilical cord mesenchyma do thin
Extracellular secrete body, dual anti-solution that FBS (hyclone) that volume fraction is 6%～7%, volume fraction are 0.6%～0.7%
The IL-3 (interleukin-3) of (penicillin/streptomycin), 4ng/mL, the SCF (stem cell factor) of 6ng/mL～7ng/mL, 6ng/mL
The IL-6 (interleukin-6) of～7ng/mL；Balance of basal medium.
In described dual anti-solution, the content of penicillin is 10000U/ml；The content of streptomysin is 10mg/ml.
In some specific embodiments, the culture medium that the present invention provides includes：Volume fraction is 33% umbilical cord mesenchyma
Dual anti-solution that FBS that stem cell excretion body, volume fraction are 6.67%, volume fraction are 0.67%, the IL-3 of 4ng/mL,
The IL-6 of SCF, 6ng/mL of 6ng/mL～7ng/mL～7ng/mL；Balance of basal medium.
The basal medium adopting in the present invention is IMDM culture medium.
The preparation method of umbilical cord mesenchymal stem cells excretion body is：By the culture supernatant of umbilical cord mesenchymal stem cells successively
After 400g centrifugation 10min, 2000g centrifugation 20min and 10000g centrifugation 30min, take supernatant, through 0.22 μm of aperture filter membrane
After filtration, 100000g is centrifuged 1h, collects resuspended with PBS after precipitation, again 100000g centrifugation 1h, gained precipitation with
PBS is resuspended, prepared umbilical cord mesenchymal stem cells excretion body.
The excretion body that the culture supernatant of every 120mL umbilical cord mesenchymal stem cells is obtained is resuspended with 2mLPBS buffer solution.
The preparation method of the culture supernatant of umbilical cord mesenchymal stem cells is：By P3～P5 for MSCs in 37 DEG C, 5%CO2
Cultivated 48 hours using serum free medium under condition of culture, collect culture supernatant.
Described serum free medium is Lonza mesenchymal stem cell serum-free culture medium (lot number：0000543297)；MSCs
Inoculum density be 1X105/mL.
Present invention also offers a kind of cultural method of procedure for peripheral blood stem cells, outside the medium culture being provided with the present invention
All candidate stem cells.
The acquisition of procedure for peripheral blood stem cells can be buied for market and alternatively voluntarily be separated or obtained by other legal means.
The separation method of procedure for peripheral blood stem cells comprises the steps：In gradient centrifugation separation peripheral blood, bone marrow mononuclear is thin
Born of the same parents.The monocyte of gained is resuspended in IMDM complete medium (IMDM+10%FBS+1% dual anti-solution+6ng/ml IL-3+
10ng/ml SCF+10ng/ml IL-6) in, density is 1 × 106Cell/mL, after culture 24h, by the peripheral blood mononuclear of gained
The method of cell magnetic bead sorting separates CD34+Candidate stem cell (Hematopoietic stem cells, HSCs).
The concrete grammar of magnetic bead sorting is：PMBC every 10 by culture7Cell is resuspended in 80 μ L magnetic beads and divides
In buffer solution, add 20 μ LCD34+Magnetic bead, mixes, and 4 DEG C are incubated 15 minutes.With unnecessary with buffer solution for cleaning after incubation
Antibody, by incubation after cell pass through Magnetic Isolation post separation, obtain CD34+Candidate stem cell.
In culture, the inoculum density of procedure for peripheral blood stem cells is 1 × 106cell/well.
The temperature of culture is 37 DEG C, saturated humidity, 5%CO2.
The invention provides the purposes of umbilical cord mesenchymal stem cells excretion body, containing umbilical cord mesenchymal stem cells excretion body
Culture medium and the cultural method of procedure for peripheral blood stem cells.Experiments indicate that, real with respect to the culture using complete medium
For testing, co-cultured with procedure for peripheral blood stem cells using umbilical cord mesenchymal stem cells excretion body, can be by the cell of culture gained
Quantity improves about 1.5 times, and Clone formation quantity also improves about 1.5 times,.Through statistical analysis, do with being not added with umbilical cord mesenchyma
The culture effect of cell excretion body is compared, and the experiment effect adding umbilical cord mesenchymal stem cells excretion body has pole significant difference (p
<0.001).
Brief description
Fig. 1 shows ESEM detection excretion body；
Fig. 2 shows excretion body nano-particle size analysis；
Fig. 3 shows HSCs cell quantity；
Fig. 4 shows HSCs body outer clone；Wherein Fig. 4-a shows experimental group HSCs body outer clone；Fig. 4-b shows that control group HSCs is external
Clone；
Fig. 5 shows HSCs body outer clone quantity；
In Fig. 3 or Fig. 5, * * * shows there is pole significant difference, p between group<0.001.
Specific embodiment
The invention provides the purposes of umbilical cord mesenchymal stem cells excretion body, those skilled in the art can use for reference interior herein
Hold, be suitably modified technological parameter and realize.Specifically, all similar replacements and change are to those skilled in the art
For be it will be apparent that they are considered as including in the present invention.Preferable enforcement has been passed through in the method for the present invention and application
Example is described, and related personnel substantially can be to methods herein and application in without departing from present invention, spirit and scope
It is modified or suitably changes and combine, to realize and to apply the technology of the present invention.
With reference to embodiment, the present invention is expanded on further：
The extraction of embodiment 1 umbilical cord MSCs excretion body separates
By well-grown P3～P5 for MSCs in 37 DEG C, 5%CO2Using serum free medium (Lonza under condition of culture
Mesenchymal stem cell serum-free culture medium) cultivate 48 hours, collect 120mL cell culture supernatant.Using ultracentrifugation method,
Sequentially pass through 400g centrifugation 10min, 2000g centrifugation 20min and 10,000g centrifugation 30min, obtain without cell, cell fragment
And the supernatant of other particulate matters.Supernatant through the membrane filtration in 0.22 μm of aperture, then passes through ultrahigh speed 100,000g from
Heart 1h, abandons supernatant and stays sediment.With the resuspended washing precipitate of 10mlPBS, 100,000g centrifugation 1h, abandons supernatant and obtains sediment again
I.e. excretion body.The centrifugal process that excretion body extracts all is carried out at 4 DEG C, excretion body appropriate (2mL) PBS resuspended rear -80 of extraction
DEG C save backup.
Obtain the identification of excretion body：
1. electron microscopic observation cellular morphology
Take 20 μ L excretion body suspensions to drip on load sample copper mesh light face, under room temperature condition, stand 1 minute, inhaled from side with filter paper
Dry liquids, dropping 20g/L phosphotungstic acid about 20 μ L on load sample copper mesh, room temperature negative staining after 1 minute filter paper blot negative staining liquid, Yu Bai
Toast under vehement lamp about 10 minutes, preservation (shown in Fig. 1) of observing under transmission electron microscope and take pictures.
Gelatinous precipitate it is seen that extracting through ultrahigh speed is verified by ESEM detection and nano-particle size analysis instrument
Vesica shape spherical in shape, its diameter mainly between 30～120nm (shown in Fig. 2), meets the morphological feature of excretion body.
2. Flow cytometry excretion body surface marker
Excretion body CD9, CD63 anti-human with the mouse that PE marks antibody at room temperature lucifuge in the MSCs source after isolating and purifying is incubated
Educate 30 minutes, washed with PBS after 1 time, the paraformaldehyde with 1% is resuspended, flow cytometer tests and analyzes.Result shows, point
Excretion body surface from MSCs source after purification reaches CD9 and CD63, meets excretion bulk properties.
Embodiment 2 human peripheral candidate stem cell isolation and purification：
Peripheral blood about 5mL, by density-gradient centrifugation method separation myelomonocyte.Concrete grammar is as follows：Bone by collection
Marrow sample adds in equivalent PBS, is slowly added to, 4 DEG C of centrifugations, and 2000rpm is centrifuged 30 minutes.Be creamy white in the middle of drawing centre
Layer, this layer is and is rich in monocytic layer.The monocyte of gained is resuspended in IMDM complete medium (IMDM+10%FBS
+ 1%PS+6ng/ml IL-3+10ng/ml SCF+10ng/ml IL-6) in, piping and druming uniformly, carries out cell count.By cell with
1x106The density of/mL is inoculated in the culture dish of 100mm, be positioned over 37 DEG C, volume fraction be 5% CO2Cultivate in incubator
24h.
The method of the PMBC magnetic bead sorting of culture gained is separated CD34+Candidate stem cell
(Hematopoietic stem cells,HSCs).Concrete grammar is as follows：By every for detached PMBC 107Resuspended
In 80 μ L Beads enrichment buffer solutions, add 20 μ LCD34+Magnetic bead, mixes, and 4 DEG C are incubated 15 minutes.With with delaying after incubation
Rush the unnecessary antibody of liquid cleaning, the cell after incubation is passed through Magnetic Isolation post separation, obtains CD34+Candidate stem cell.
Embodiment 3
Experimental group：
The HSCs that embodiment 2 is obtained is with 1 × 106Cell/mL density is inoculated in culture medium 1mL (IMDM+10% in 24 orifice plates
FBS+1%PS+6ng/ml IL-3+10ng/ml SCF+10ng/ml IL-6), add 0.5mL umbilical cord MSCs excretion body, culture
Base and excretion body volume ratio are 2:1, it is placed in 5%CO237 DEG C of cultures of incubator, with 1 after 3 days:1 volume ratio and 0.4% platform
Expect that indigo plant mixes, be placed in basis of microscopic observation and count, calculate cell viability according to staining conditions.
Cell after culture is with 6 × 103Density be inoculated in 0.3mL IMDM complete medium, be transferred to
MethocultTMIn H4531 culture medium, mix that rear chamber is gentle and quiet puts 5 minutes.1.1mL cell and culture medium suspension are transferred to
In 35mm Tissue Culture Dish, it is placed in 5%CO237 DEG C of cultures of incubator, observed clonality after 7 days, and in optical microphotograph
Take pictures under mirror counting.
Control group：
With the cell without excretion body for comparison, the HSCs that specially embodiment 2 is obtained is with 1 × 106Cell/mL density
It is inoculated in (the IMDM+10%FBS+1%PS+6ng/ml IL-3+10ng/ml SCF+10ng/ml of culture medium 1mL in 24 orifice plates
IL-6), after cultivating 6 days, with 1:1 volume ratio is mixed with 0.4% trypan blue, is placed in basis of microscopic observation and counts,
Calculate cell viability according to staining conditions.
Cell after culture is with 6 × 103Density be inoculated in 0.3mL IMDM complete medium, be transferred to
MethocultTMIn H4531 culture medium, mix that rear chamber is gentle and quiet puts 5 minutes.1.1mL cell and culture medium suspension are transferred to
In 35mm Tissue Culture Dish, it is placed in 5%CO237 DEG C of cultures of incubator, observed clonality after 7 days, and in optical microphotograph
Take pictures under mirror counting.
Cell counts such as Fig. 3, according to Fig. 3, adding excretion body can make cell quantity improve 1.5 times about, and right
Photograph ratio, this effect has pole significant difference (p<0.001).
Cell clonal formation situation such as Fig. 4-a (experimental group) and Fig. 4-b (control group), clone number statistics such as Fig. 5, knot
Fruit shows, adds excretion body and extremely can significantly improve Cell clonality (p<0.001).
The above is only the preferred embodiment of the present invention it is noted that coming for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (9)
1. application in improving procedure for peripheral blood stem cells clonality for the umbilical cord mesenchymal stem cells excretion body.
2. application in promoting procedure for peripheral blood stem cells propagation for the umbilical cord mesenchymal stem cells excretion body.
3. a kind of culture medium of procedure for peripheral blood stem cells is it is characterised in that contain umbilical cord mesenchymal stem cells excretion body.
4. culture medium according to claim 3 is it is characterised in that include：Volume fraction is between 30%～35% umbilical cord
Dual anti-solution that FBS that mesenchymal stem cells excretion body, volume fraction are 6%～7%, volume fraction are 0.6%～0.7%,
The IL-6 of SCF, 6ng/mL of IL-3,6ng/mL of 4ng/mL～7ng/mL～7ng/mL；Balance of basal medium.
5. culture medium according to claim 3 is it is characterised in that the preparation side of described umbilical cord mesenchymal stem cells excretion body
Method is：By the culture supernatant of umbilical cord mesenchymal stem cells sequentially pass through 400g centrifugation 10min, 2000g centrifugation 20min and
After 10000g centrifugation 30min, take supernatant, after 0.22 μm of aperture membrane filtration, 100000g is centrifuged 1h, collect after precipitation with
PBS is resuspended, again 100000g centrifugation 1h, and gained precipitates, prepared umbilical cord mesenchymal stem cells resuspended with PBS
Excretion body.
6. culture medium according to claim 3 is it is characterised in that described basal medium is IMDM culture medium.
7. a kind of cultural method of procedure for peripheral blood stem cells is it is characterised in that with the culture described in any one of claim 3～6
Procedure for peripheral blood stem cells cultivated by base.
8. cultural method according to claim 7 is it is characterised in that in described culture, the inoculation of procedure for peripheral blood stem cells
Density is 1 × 106cell/well.
9. cultural method according to claim 7 it is characterised in that described culture temperature be 37 DEG C, saturated humidity,
5%CO2.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446891A (en) * | 2017-09-30 | 2017-12-08 | 吉林省太阳鸟再生医学工程有限责任公司 | A kind of method that human umbilical cord's blood candidate stem cell is expanded using itself umbilical cord mesenchymal stem cells as stroma cell |
| CN109182259A (en) * | 2018-08-29 | 2019-01-11 | 浙江大学 | A method of improving mescenchymal stem cell excretion body yield |
| CN110339147A (en) * | 2018-11-27 | 2019-10-18 | 浙江梵泊细胞工程有限公司 | A kind of composition for external application and application thereof |
| CN110643570A (en) * | 2019-09-29 | 2020-01-03 | 华夏源(上海)生命科技有限公司 | Preparation method of ESO (ethylene-vinyl acetate copolymer) multi-factor |
| CN110804607A (en) * | 2019-11-18 | 2020-02-18 | 深圳市润科生物科技有限公司 | Preparation method of high-concentration human mesenchymal stem cell lysate |
| CN111358810A (en) * | 2020-02-28 | 2020-07-03 | 广州市天河诺亚生物工程有限公司 | Compound for assisting anemia treatment and preparation method thereof |
| CN111394307A (en) * | 2020-02-17 | 2020-07-10 | 天津医科大学眼科医院 | Method for separating and purifying exosome from plasma and application |
| CN112280734A (en) * | 2018-05-17 | 2021-01-29 | 广东芙金干细胞再生医学有限公司 | Preparation method of exosome and stem cell proliferation reagent containing exosome |
| CN113736730A (en) * | 2021-09-09 | 2021-12-03 | 天晴干细胞股份有限公司 | Method for culturing umbilical cord tissue mesenchymal cells |
| CN114159550A (en) * | 2021-11-16 | 2022-03-11 | 南京天纵易康生物科技股份有限公司 | Preparation method of microcapsule hair-growing product based on exosome growth factor |
| WO2022217868A1 (en) * | 2021-04-13 | 2022-10-20 | 广东唯泰生物科技有限公司 | Application of exosome in promoting growth of parietal decidual basalis‑mesenchymal stem cells |
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- 2016-09-30 CN CN201610882018.8A patent/CN106399247A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107446891A (en) * | 2017-09-30 | 2017-12-08 | 吉林省太阳鸟再生医学工程有限责任公司 | A kind of method that human umbilical cord's blood candidate stem cell is expanded using itself umbilical cord mesenchymal stem cells as stroma cell |
| CN107446891B (en) * | 2017-09-30 | 2019-01-18 | 吉林省太阳鸟再生医学工程有限责任公司 | A method of expanding human umbilical cord's blood candidate stem cell using itself umbilical cord mesenchymal stem cells as stroma cell |
| CN112280734A (en) * | 2018-05-17 | 2021-01-29 | 广东芙金干细胞再生医学有限公司 | Preparation method of exosome and stem cell proliferation reagent containing exosome |
| CN109182259A (en) * | 2018-08-29 | 2019-01-11 | 浙江大学 | A method of improving mescenchymal stem cell excretion body yield |
| CN110339147A (en) * | 2018-11-27 | 2019-10-18 | 浙江梵泊细胞工程有限公司 | A kind of composition for external application and application thereof |
| CN110643570A (en) * | 2019-09-29 | 2020-01-03 | 华夏源(上海)生命科技有限公司 | Preparation method of ESO (ethylene-vinyl acetate copolymer) multi-factor |
| CN110804607A (en) * | 2019-11-18 | 2020-02-18 | 深圳市润科生物科技有限公司 | Preparation method of high-concentration human mesenchymal stem cell lysate |
| CN111394307A (en) * | 2020-02-17 | 2020-07-10 | 天津医科大学眼科医院 | Method for separating and purifying exosome from plasma and application |
| CN111358810A (en) * | 2020-02-28 | 2020-07-03 | 广州市天河诺亚生物工程有限公司 | Compound for assisting anemia treatment and preparation method thereof |
| WO2022217868A1 (en) * | 2021-04-13 | 2022-10-20 | 广东唯泰生物科技有限公司 | Application of exosome in promoting growth of parietal decidual basalis‑mesenchymal stem cells |
| CN113736730A (en) * | 2021-09-09 | 2021-12-03 | 天晴干细胞股份有限公司 | Method for culturing umbilical cord tissue mesenchymal cells |
| CN114159550A (en) * | 2021-11-16 | 2022-03-11 | 南京天纵易康生物科技股份有限公司 | Preparation method of microcapsule hair-growing product based on exosome growth factor |
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Application publication date: 20170215 |