CN110205290A - A kind of preparation method of placenta mesenchyma stem cell microcapsule powder - Google Patents
A kind of preparation method of placenta mesenchyma stem cell microcapsule powder Download PDFInfo
- Publication number
- CN110205290A CN110205290A CN201910402460.XA CN201910402460A CN110205290A CN 110205290 A CN110205290 A CN 110205290A CN 201910402460 A CN201910402460 A CN 201910402460A CN 110205290 A CN110205290 A CN 110205290A
- Authority
- CN
- China
- Prior art keywords
- cell
- stem cell
- added
- culture
- placenta
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Rheumatology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of preparation methods of placenta mesenchyma stem cell microcapsule powder, it is comprised the following steps: one, the tissue in clip placenta decidua basalis face is cut into muddy flesh, is put into born of the same parents' culture bottle and carries out originally culture；Two, cell suspending liquid is taken to be centrifuged 5min into new centrifuge tube；Lower sediment is collected, primary culture medium is added；Three, freezing-thawing and cracking obtains cell mixture after cell growth, by cell mixture centrifugal treating；Four, take supernatant in new sterile centrifugation tube, cryo-conservation；Five, 5% Arabic gum wall material solution is prepared；Six, 1% chitosan wall material solution is prepared；Prepare w/o type emulsion；Seven, W/O/W type emulsion is prepared；Eight, complex coacervation；Nine, curing reaction；Ten, freeze-drying obtains placenta mesenchyma stem cell microcapsules；Active material is prepared into microcapsules and protected by the present invention, is reduced the influences of the factors to active material such as temperature, illumination, is reached the requirement of normal temperature storage.
Description
Technical field
The present invention relates to cell preparation and skin care technical fields, and in particular to a kind of placenta mesenchyma stem cell microcapsule powder
Preparation method.
Background technique
2007, British scientist Anastasia write articles on Nature magazine and points out: adult stem cell to human body self
The main reason for reparation and regeneration are most important, and the reduction of adult stem cell is human senility.The mankind are managed in this discovery
Solution aging course and stem cell transplantation are of great significance, this is also that also regenerative medicine research flourishes stem cell in recent years
The reason of.
Mescenchymal stem cell (MSC) is derived from the mesoderm and ectoderm of mesoderm growing early stage.Mescenchymal stem cell is that one kind has
The adult stem cell of the of self-replication capacity and multi-lineage potential belongs to non-terminal differentiation, its existing interstitial cell, and has endothelium
The feature of cell and epithelial cell.In the normal tissue damage repair process of body, MSC be a kind of important participation tissue again
Raw cell bank.Under the effect of the distinctive signal caused by tissue damage, MSC moves to damaged part, is proliferated in localized clusters, according to
Break up according to different damage signals along different approaches.MSC is easily isolated amplification, and amplification in vitro ability is strong, even if amplification is several
It is still able to maintain its Multidirectional Differentiation ability again.Therefore, MSC is a kind of practical tissue repair seed cell.
For mescenchymal stem cell as a kind of multipotential stem cell, the physics that cell growth can be both provided for candidate stem cell is micro-
Environment, and a large amount of cell factor can be secreted, such as stem cell factor (Stem Cell Growth Factors, SCF), blood is small
Plate generates element, and blood vessel production element, interleukin-13, interleukin 6 etc., supply stem cells hyperplasia and differentiation use.Clinical trial is by the U.S.
The clinical trial that Massachusetts Polytechnics, Ha Fu university carries out has 147 appearance apparent with control group in 156 SCF subjects
Difference, it was demonstrated that SCF can be improved memory, delay senescence.
Compared to other stem cells, placenta mesenchyma stem cell (placental mesenchymal stem cells,
PMSCs) there are convenient sources, cell quantity is sufficient, is easily isolated, cultivates, expands and purifies, and passage amplification still has after more than 30 generations
There are stem cell properties.It is a kind of multipotential stem cell extracted from placenta tissue, is clinically had broad application prospects, including
The diseases such as acute liver damage, diabetes, lower limb ischemia, preeclampsia, autoimmune disease, osteoarthritis, early ageing disease are controlled
It treats.Current study show that placenta is the best source of mescenchymal stem cell.
For mescenchymal stem cell in addition to the adjuvant treatment for disease, anti-aging is also the Xiang Chong that scientists are being studied
Direction is wanted, skin is the maximum organ of human body area, and skin, as the age is continuously increased, all gradually becomes as other organs
To aging.There are many reason of causing skin aging, such as oxidative damage (ROS damage): active oxygen (reactive oxygen
Species, ROS) and active nitrogen (reactive nitrogen species, RNS) be cause protein oxidative damage important
Factor.In the cell and in external environment, protein is the main target of free radical and the effect of other oxidants.It is estimated that thin
In macromolecular intracellular, by the 50%-75% for the free fiduciary point living radical total amount that protein is removed.Due to certain protein
With longer half-life period, it is be easy to cause the accumulation of oxidative damage, accelerates skin aging process.
MSC can discharge the proliferation and differentiation for promoting the growth factor such as SCF of cell growth to promote cell, while MSC
A kind of chemical substance NADH (Nicotinamide adenine dinucleotide) can also be discharged, is nicotinamide adenine
The reduction-state of dinucleotides, reduced coenzyme Ⅰ is the direct sources substance of energy needed for cell is grown, and participates on human body
Thousand kinds or more of metabolic response, without NADH, cell is unable to maintain that normal structure and function.NADH content is higher in cell,
Energy caused by it is more, and the function of this cell is better, and the service life of the cell and entire individual is longer.NADH
In human body, there are four types of existence form (NAD+/NADH and NADP+/NADPH), and NADH itself is a kind of very strong biological antioxidant
Agent can effectively remove human body harmful free radicals.NADPH can promote other lifes of human body with human activin antioxidation system
The regeneration (such as vitamin C, vitamin E, glutathione) of object antioxidant.NAD+ is small point for capable of really going deep into cell tissue
Son effectively activates the activity of longevity gene Sirt1~7, and fundamentally activation inhibits aging gene growth, delays senescence.
Therefore, MSC is just increasingly being used for the anti-ageing research and application of skin.Existing cell class active material, it is generally existing
Property is unstable, especially stringent to temperature requirement vulnerable to the influence of the environmental factors such as temperature, illumination, if be not protected,
It is easy to inactivate under normal temperature condition.
Summary of the invention
In view of the defects and deficiencies of the prior art, the present invention intends to provide a kind of placenta mesenchyma stem cell micro-capsules
Active material is prepared into microcapsules and protected by the preparation method of powder, reduces the factors such as temperature, illumination to the shadow of active material
It rings, reaches the requirement of normal temperature storage.
To achieve the above object, the technical solution adopted by the present invention is that: it is comprised the following steps:
One, the placenta discoidalis of full-term normal delivery or Cesarean esction, the tissue in clip placenta decidua basalis face are chosen；
Two, the tissue vivid in desinfection chamber picking color, is cut into muddy flesh；
Three, the tissue shredded is laid in the Tissue Culture Flask of T75cm2 with cell scraper, is marked；
Four, Tissue Culture Flask is buckled to, is slowly added to 6mL serum free medium, tightens bottle cap, be put into 37 DEG C, 5%
In CO2 incubator, it is inverted 2h；
Five, Tissue Culture Flask is just being set, it is ensured that culture solution can cover every piece of tissue, stationary culture；
Six, tissue block is observed after three days, movement is light when observation, try not to shake, cell is waited to climb out of；
Seven, half amount changes liquid again after seven days, and hereafter half amount changes liquid；
Eight, when cell confluency degree reaches 80% or so, originally culture is carried out；
Nine, by broth out, normal saline flushing one time, the 0.25%Trypsin-EDTA (1X) of 2mL is added, is put into
In 37 DEG C of incubators, about 3-5min stops digestion when cell rounding, discards digestive juice, and serum free medium is added to culture
Bottle terminates digestion, gently blows and beats adherent cell, is uniformly distributed in cell in solution, draws cell suspending liquid to new centrifugation
Guan Zhong is centrifuged 5min, revolving speed 1500rpm；
Ten, upper solution is discarded, lower sediment is collected, primary culture medium is added, is carried out according to 1 × 105/mL cell concentration
Originally culture；
11, when cell grows to 80-90% density, cell is harvested；It counts, takes 1 × 108 placenta mesenchyma dry thin
The 50mL sterile deionized water of pre-cooling is added in born of the same parents, and multigelation cracks cell sufficiently, freezing in other cell liquid nitrogen；
12, several milliliters of cells frozen storing liquid is added in 50mL centrifuge tube and is resuspended for the cell number frozen as needed
Cell suspension is made in cell；
13, cell suspension is moved into the cryopreservation tube of several 2mL, every pipe adds cell suspension 1mL, by cryopreservation tube lid
It tightens and covers；
14, cryopreservation tube is placed in the program temperature reduction box for having equilibrated to 4 DEG C, moves to -80 DEG C of refrigerators and cools down -80 step by step
℃；
15, cryopreservation tube is taken out from program temperature reduction box, move to rapidly in liquid nitrogen freeze for a long time it is spare；
16, the cell mixture after cracking in the 11st step is taken, is in 4 DEG C of at a temperature of centrifugation 10min1 revolving speed
10000rpm；The supernatant containing active material is drawn in new sterile centrifugation tube, is settled to 100mL, cryo-conservation is spare；
17,5g Arabic gum and 100g deionized water are weighed, is dissolved in 60 DEG C of stirred in water bath, 5% Arab is obtained
Glue wall material solution；
18,1g chitosan and 100g deionized water are weighed, is dissolved in 50 DEG C of stirred in water bath, obtains 1% chitosan wall
Material solution；
19, the supernatant of cryo-conservation in step 10 six is added in peanut oil, the volume ratio of supernatant and peanut oil
For 1:3,40min, revolving speed 10000rpm are emulsified in 35 DEG C of at a temperature of high speed dispersion, obtains uniform w/o type emulsion；
20, w/o type emulsion is added in 5% Arabic gum, w/o type emulsion and the volume of 5% Arabic gum are
Than 1:4, it is 10000rpm in 35 DEG C of at a temperature of high speed dispersion emulsification 30min1 revolving speed, obtains uniform W/O/W type emulsion；
21, above-mentioned W/O/W type emulsion is added in 1% chitosan；W/O/W type emulsion and 1% chitosan
Volume ratio is 1:1, under constant temperature at the uniform velocity stirring condition, adjusts PH to 4-5 with 1mol/LHCl solution and 0.1g/mLNaOH；At 25 DEG C
At a temperature of high speed dispersion emulsify 30min, revolving speed 10000rpm, make Arabic gum and chitosan that multiple cohesion occur；
22, under room temperature, solidify 4h；
23, vacuum freeze drier starts to vacuumize after being cooled to -45 DEG C in advance, controls in 80Kpa, cooling time 8h,
Obtain placenta mesenchyma stem cell microcapsules.
Further, the serum free medium in step 4 and step 9 includes- XF human mesenchyme is dry thin
Born of the same parents' basal medium and additive, and be sufficiently mixed,- XF human mesenchymal stem cell basal medium and additive
It is sufficiently mixed with the volume ratio of 10:1, L-gln is added after mixing, its final concentration is made to reach 2mM；
Further, the primary culture medium in step 10 includes complete medium- XF and MSCGM-
CDTMBulletKit；Complete medium- XF and MSCGM-CDTMThe mass ratio of BulletKit is 3:1；
Further, in step 12, the additive amount of cells frozen storing liquid is that 1ml frozen stock solution is added in 1 × 107 cell；
It further, include following raw material: human serum albumin 50%, nothing in cells frozen storing liquid in step 12
Serum free culture system liquid 40%, dimethyl sulfoxide 10%；
Further, in step 10 seven, No. CAS of Arabic gum is 9000-01-5；
Further, in step 10 eight, No. CAS of chitosan is 9012-76-4；
Further, in step 2 11, No. CAS of 1mol/LHCl solution is 7647-01-0；0.1g/mLNaOH's
No. CAS is 1310-73-2.
After adopting the above scheme, the invention has the following beneficial effects: a kind of placenta mesenchyma stem cell micro-capsule of the present invention
Active material is prepared into microcapsules and protected by the preparation method of powder for the anti-ageing research and application of skin, reduces temperature
Influence of the factors such as degree, illumination to active material, reaches the requirement of normal temperature storage.
Specific embodiment
The present invention is further illustrated below.
Present embodiment the technical solution adopted is that: it is comprised the following steps:
One, the placenta discoidalis of full-term normal delivery or Cesarean esction, the tissue in clip placenta decidua basalis face are chosen；
Two, the tissue vivid in desinfection chamber picking color, is cut into muddy flesh；
Three, the tissue shredded is laid in T75cm with cell scraper2Tissue Culture Flask in, mark；
Four, Tissue Culture Flask is buckled to, is slowly added to serum free medium, be put into 37 DEG C of incubators, tighten bottle cap, put
Enter 37 DEG C, in 5% CO2 incubator, is inverted culture 2h；
Five, Tissue Culture Flask is just being set, it is ensured that culture solution can cover every piece of tissue, stationary culture；
Six, tissue block is observed after three days, movement is light when observation, try not to shake；
Seven, half amount changes liquid again after seven days, and hereafter half amount changes liquid；
Eight, when cell confluency degree reaches 80% or so, originally culture is carried out；
Nine, by broth out, normal saline flushing one time, the 0.25%Trypsin-EDTA (1X) of 2mL is added, is put into
In 37 DEG C of incubators, about 3-5min stops digestion when cell rounding, discards digestive juice, and serum free medium is added to culture
Bottle terminates digestion, gently blows and beats adherent cell, is uniformly distributed in cell in solution, draws cell suspending liquid to new centrifugation
Guan Zhong is centrifuged 5min, revolving speed 1500rpm；
Ten, upper solution is discarded, lower sediment is collected, primary culture medium is added, is carried out according to 1 × 105/mL cell concentration
Originally culture；Primary culture medium includes complete medium- XF and MSCGM-CDTMBulletKit；Complete medium- XF and MSCGM-CDTMThe mass ratio of BulletKit is 3:1；
11, when cell grows to 80-90% density, cell is harvested；It counts, takes 1 × 108 placenta mesenchyma dry thin
The 50mL sterile deionized water of pre-cooling is added in born of the same parents, and multigelation cracks cell sufficiently, freezing in other cell liquid nitrogen；
12, several milliliters of cells frozen storing liquid is added in 50mL centrifuge tube and is resuspended for the cell number frozen as needed
Cell is prepared into cell suspension；The additive amount of cells frozen storing liquid is that 1ml frozen stock solution is added in 1 × 107 cell；
13, cell suspension is moved into the cryopreservation tube of several 2mL, every pipe adds cell suspension 1mL, by cryopreservation tube lid
It tightens and covers；
14, cryopreservation tube is placed in the program temperature reduction box for having equilibrated to 4 DEG C, moves to -80 DEG C of refrigerators and cools down -80 step by step
℃；
15, cryopreservation tube is taken out from program temperature reduction box, move to rapidly in liquid nitrogen freeze for a long time it is spare；
16, the cell mixture after cracking in the 11st step is taken, is in 4 DEG C of at a temperature of centrifugation 10min1 revolving speed
10000rpm；The supernatant containing active material is drawn in new sterile centrifugation tube, is settled to 100mL, cryo-conservation is spare；
17,5g Arabic gum and 100g deionized water are weighed, is dissolved in 60 DEG C of stirred in water bath, 5% Arab is obtained
Glue wall material solution；Arabic gum No. CAS: 9000-01-5；
18,1g chitosan and 100g deionized water are weighed, is dissolved in 50 DEG C of stirred in water bath, obtains 1% chitosan wall
Material solution；No. CAS: 9012-76-4 of chitosan；
19, the supernatant of cryo-conservation in step 10 six is added in peanut oil, the volume ratio of supernatant and peanut oil
For 1:3,40min, revolving speed 10000rpm are emulsified in 35 DEG C of at a temperature of high speed dispersion, obtains uniform w/o type emulsion；
20, w/o type emulsion is added in 5% Arabic gum, w/o type emulsion and the volume of 5% Arabic gum are
Than 1:4, it is 10000rpm in 35 DEG C of at a temperature of high speed dispersion emulsification 30min1 revolving speed, obtains uniform W/O/W type emulsion；
21, above-mentioned W/O/W type emulsion is added in 1% chitosan；W/O/W type emulsion and 1% chitosan
Volume ratio is 1:1, under constant temperature at the uniform velocity stirring condition, adjusts PH to 4-5 with 1mol/LHCl solution and 0.1g/mLNaOH；1mol/
No. CAS of LHCl solution is 7647-01-0；No. CAS of 0.1g/mLNaOH is 1310-73-2；25 DEG C at a temperature of divide at a high speed
Emulsification 30min, revolving speed 10000rpm are dissipated, makes Arabic gum and chitosan that multiple cohesion occur；22, room temperature condition
Under, solidify 4h；
23, vacuum freeze drier starts to vacuumize after being cooled to -45 DEG C in advance, controls in 80Kpa, cooling time 8h,
Obtain placenta mesenchyma stem cell microcapsules.
In present embodiment, the serum free medium in step 4 and step 9 includesThe human world-XF
Mesenchymal stem cells basal medium and additive, and be sufficiently mixed,- XF human mesenchymal stem cell basal medium
It is sufficiently mixed with additive with the volume ratio of 10:1, L-gln is added after mixing, its final concentration is made to reach 2mM；
In step 4, Tissue Culture Flask back-off after, the culture medium of certain volume can be accommodated because culture bottle bottleneck with
Body has certain angle, and the culture medium of certain volume can be accommodated by the angle of bottleneck, and Tissue Culture Flask back-off can be prevented
The only culture overdrying of top is unfavorable for normally climbing out of for cell；It can also prevent from taking out culture bottle from incubator
It is normally adherent that cell is influenced again plus when culture medium, or even causes secondary pollution；
Existing cell class active material, generally existing property is unstable, the shadow vulnerable to environmental factors such as temperature, illumination
It rings, it is especially stringent to temperature requirement, if be not protected, it is easy to inactivate under normal temperature condition.Existing active material is equal
Cryo-conservation, if long-distance transport, it is necessary to carry out at low temperature, need cold chain transportation, cold chain transportation is extremely inconvenient.This specific embodiment party
The preparation method of placenta mesenchyma stem cell microcapsule powder, prepares cell active substance described in formula, active material is prepared into micro-
Capsule is protected, and is reduced the influences of the factors to active material such as temperature, illumination, is reached the requirement of normal temperature storage, and skin is used for
Anti-ageing research and application.
The above is merely illustrative of the technical solution of the present invention, rather than limits those of ordinary skill in the art to this hair
The other modifications or equivalent replacement that bright technical solution is made, as long as it does not depart from the spirit and scope of the technical scheme of the present invention,
It is intended to be within the scope of the claims of the invention.
Claims (8)
1. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder, it is characterised in that it is comprised the following steps:
One, the placenta discoidalis of full-term normal delivery or Cesarean esction, the tissue in clip placenta decidua basalis face are chosen；
Two, the tissue vivid in desinfection chamber picking color, is cut into muddy flesh；
Three, the tissue shredded is laid in the Tissue Culture Flask of T75cm2 with cell scraper, is marked；
Four, Tissue Culture Flask is buckled to, is slowly added to 6ml serum free medium, tightens bottle cap, be put into 37 DEG C of incubators, to
It is filled with 5% CO2 in incubator, is inverted culture 2h, tightens bottle cap；
Five, Tissue Culture Flask is just being set, it is ensured that culture solution can cover every piece of tissue, stationary culture；
Six, tissue block is observed after three days, movement is light when observation, try not to shake, cell is waited to climb out of；
Seven, half amount changes liquid again after seven days, and hereafter half amount changes liquid；
Eight, when cell confluency degree reaches 80% or so, originally culture is carried out；
Nine, by broth out, normal saline flushing one time, the 0.25%Trypsin-EDTA (1X) of 2mL is added, is put into 37 DEG C
In incubator, about 3-5min stops digestion when cell rounding, discards digestive juice, and serum free medium is added to culture bottle end
It only digests, gently blows and beats adherent cell, be uniformly distributed in cell in solution, draw cell suspending liquid to new centrifuge tube
In, it is centrifuged 5min, revolving speed 1500rpm；
Ten, upper solution is discarded, lower sediment is collected, primary culture medium is added, is carried out according to 1 × 105/mL cell concentration primary
Culture；
11, when cell grows to 80-90% density, cell is harvested；It counts, takes 1 × 108 placenta mesenchyma stem cell, add
Enter the 50mL sterile deionized water of pre-cooling, multigelation cracks cell sufficiently, freezing in other cell liquid nitrogen；
12, the cell number frozen as needed several milliliters of cells frozen storing liquid is added in 50mL centrifuge tube, cell is resuspended,
Cell suspension is made；
13, cell suspension is moved into the cryopreservation tube of several 2mL, every pipe adds cell suspension 1mL, and cryopreservation tube lid is tightened
It covers；
14, cryopreservation tube is placed in the program temperature reduction box for having equilibrated to 4 DEG C, moves to -80 DEG C of refrigerators and cools down -80 DEG C step by step；
15, cryopreservation tube is taken out from program temperature reduction box, move to rapidly in liquid nitrogen freeze for a long time it is spare；
16, the cell mixture after cracking in the 11st step is taken, is in 4 DEG C of at a temperature of centrifugation 10min1 revolving speed
10000rpm；The supernatant containing active material is drawn in new sterile centrifugation tube, is settled to 100mL, cryo-conservation is spare；
17,5g Arabic gum and 100g deionized water are weighed, is dissolved in 60 DEG C of stirred in water bath, obtains 5% Arabic gum wall
Material solution；
18,1g chitosan and 100g deionized water are weighed, is dissolved in 50 DEG C of stirred in water bath, it is molten to obtain 1% chitosan wall material
Liquid；
19, the supernatant of cryo-conservation in step 10 six is added in peanut oil, the volume ratio of supernatant and peanut oil is 1:
3,40min, revolving speed 10000rpm are emulsified in 35 DEG C of at a temperature of high speed dispersion, obtains uniform w/o type emulsion；
20, w/o type emulsion is added in 5% Arabic gum, the volume of w/o type emulsion and 5% Arabic gum is than 1:
4, it is 10000rpm in 35 DEG C of at a temperature of high speed dispersion emulsification 30min1 revolving speed, obtains uniform W/O/W type emulsion；
21, above-mentioned W/O/W type emulsion is added in 1% chitosan；The volume of W/O/W type emulsion and 1% chitosan
Than under constant temperature at the uniform velocity stirring condition, adjusting PH to 4-5 with 1mol/LHCl solution and 0.1g/mLNaOH for 1:1；In 25 DEG C of temperature
It spends lower high speed dispersion and emulsifies 30min, revolving speed 10000rpm, make Arabic gum and chitosan that multiple cohesion occur；
22, under room temperature, solidify 4h；
23, vacuum freeze drier starts to vacuumize after being cooled to -45 DEG C in advance, and control is obtained in 80Kpa, cooling time 8h
Placenta mesenchyma stem cell microcapsules.
2. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder according to claim 1, it is characterised in that step
Four and step 9 in serum free medium include- XF human mesenchymal stem cell basal medium and additive,
And be sufficiently mixed,- XF human mesenchymal stem cell basal medium and additive are sufficiently mixed with the volume ratio of 10:1
It closes, L-gln is added after mixing, its final concentration is made to reach 2mM.
3. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder according to claim 1, it is characterised in that step
Primary culture medium in ten includes complete medium- XF and MSCGM-CDTMBulletKit；Complete medium- XF and MSCGM-CDTMThe mass ratio of BulletKit is 3:1.
4. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder according to claim 1, it is characterised in that step
In 12, the additive amount of cells frozen storing liquid is that 1ml frozen stock solution is added in 1 × 107 cell.
5. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder according to claim 1 or 4, it is characterised in that step
It include following raw material: human serum albumin 50%, serum-free medium 40%, diformazan in cells frozen storing liquid in rapid 12
Base sulfoxide 10%.
6. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder according to claim 1, it is characterised in that step
In 17, No. CAS of Arabic gum is 9000-01-5.
7. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder according to claim 1, it is characterised in that step
In 18, No. CAS of chitosan is 9012-76-4.
8. a kind of preparation method of placenta mesenchyma stem cell microcapsule powder according to claim 1, it is characterised in that step
In 21, No. CAS of 1mol/LHCl solution is 7647-01-0；No. CAS of 0.1g/mLNaOH is 1310-73-2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910402460.XA CN110205290A (en) | 2019-05-15 | 2019-05-15 | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910402460.XA CN110205290A (en) | 2019-05-15 | 2019-05-15 | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110205290A true CN110205290A (en) | 2019-09-06 |
Family
ID=67787246
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910402460.XA Pending CN110205290A (en) | 2019-05-15 | 2019-05-15 | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110205290A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103009A (en) * | 2018-01-31 | 2018-06-01 | 溯源生命科技股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell |
| CN109223704A (en) * | 2018-08-17 | 2019-01-18 | 溯源生命科技股份有限公司 | A kind of preparation method of mescenchymal stem cell nano essence |
| CN109619561A (en) * | 2018-12-28 | 2019-04-16 | 溯源生命科技股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder |
-
2019
- 2019-05-15 CN CN201910402460.XA patent/CN110205290A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103009A (en) * | 2018-01-31 | 2018-06-01 | 溯源生命科技股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell |
| CN109223704A (en) * | 2018-08-17 | 2019-01-18 | 溯源生命科技股份有限公司 | A kind of preparation method of mescenchymal stem cell nano essence |
| CN109619561A (en) * | 2018-12-28 | 2019-04-16 | 溯源生命科技股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jang et al. | Cryopreservation and its clinical applications | |
| Beers et al. | Ovarian plasminogen activator: relationship to ovulation and hormonal regulation | |
| CN109090100A (en) | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method | |
| EP2998389A1 (en) | Production method for sheet-shaped cell culture | |
| CN107810948B (en) | Serum-free freezing medium for human umbilical cord mesenchymal stem cells | |
| CN107306936B (en) | Method for preserving and transporting stem cells under normal temperature condition and matrix used by method | |
| JP2010539970A5 (en) | ||
| KR20100087705A (en) | Optimised and defined method for isolation and preservation of precursor cells from human umbilical cord | |
| WO2013154404A1 (en) | Method and composition for preventing stem cell disruption and aggregation | |
| CN114557337B (en) | Protein-free non-program freezing solution of umbilical cord mesenchymal stem cells and preparation method thereof | |
| CN111494420A (en) | Human umbilical cord mesenchymal stem cell injection, preparation method and application thereof | |
| CN108184818A (en) | A kind of Human plactnta mesenchyma stem cell suspension protective agent | |
| EP3530729A1 (en) | Preservative solution for live cells or composition comprising live cells | |
| CN106520679A (en) | Kit for culturing pancreas stem cells | |
| EP3527077A1 (en) | Preservative solution for live cells or composition comprising live cells | |
| CN109619561A (en) | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder | |
| CN112167241A (en) | Stem cell freezing medium and stem cell freezing and recovering method | |
| CN108753712B (en) | Method for extracting adipose-derived stem cells | |
| CN110205290A (en) | A kind of preparation method of placenta mesenchyma stem cell microcapsule powder | |
| CN110199984A (en) | A kind of umbilical cord mesenchymal stem cells frozen stock solution and application thereof | |
| CN109576308A (en) | A kind of method and its application improving human stem cells source hepatic lineage function of detoxification | |
| CN112655700B (en) | Application of frozen stock solution in gallbladder stem cells and recovery method of gallbladder stem cells | |
| RU2331670C1 (en) | Method of accelerated acquiring of mesenchymal stem cells (msc) culture of mammals, except human, with low heterogeneity and high viability | |
| CN113632784A (en) | Stem cell cryopreservation protective agent and application method thereof | |
| Globa et al. | Contractile activity of detrusor of rats with infravesical obstruction after introduced cryoextract of spinal ganglia and biologically active products of mantle gliocyte culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190906 |