CN101914490A - Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof - Google Patents
Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof Download PDFInfo
- Publication number
- CN101914490A CN101914490A CN 201010252201 CN201010252201A CN101914490A CN 101914490 A CN101914490 A CN 101914490A CN 201010252201 CN201010252201 CN 201010252201 CN 201010252201 A CN201010252201 A CN 201010252201A CN 101914490 A CN101914490 A CN 101914490A
- Authority
- CN
- China
- Prior art keywords
- cell
- human
- mesenchymal stem
- stem cell
- serum
- 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.)
- Granted
Links
Images
Abstract
The invention relates to a human amnion mesenchymal stem cell serum-free culture medium and a culture method thereof. The culture medium is formed by adding human serum albumin, human transferrin, human insulin and sodium selenite into a DMEM/F12 basic culture medium. The culture method for the culture medium comprises the following steps of: digesting human amnion by using trypsin, then digesting the human amnion by using collagenase IV and deoxyribonuclease I, and filtering the mixture to obtain single cell suspension; and adding the human serum albumin, the transferrin, the insulin and the sodium selenite into the DMEM/F12 basic culture medium in a ratio of VDMEM to VF12 of 1:1, and putting human amnion mesenchymal stem cells in a 37 DEG C CO2 incubator with saturated humidity and volume fraction of 5 percent under the serum-free condition, wherein culture in vitro and amplification are realized by solution change and transfer of culture, potentiality of multi-direction differentiation is maintained, and the amplified cells can be induced in vitro to form cartilage cells, osteoblasts and adipocytes. The culture medium and the culture method have the characteristics of no other animal sources, wide source and no limitation of ethics.
Description
Technical field
The invention belongs to a kind of mescenchymal stem cell substratum and the cultural method thereof of biological technical field, what be specifically related to is a kind of human amnion mesenchymal stem cell serum-free culture medium and cultural method thereof.
Background technology
In the last few years, regenerative medicine was flourish, and the research of stem cell has obtained important breakthrough. and use stem cell to generate body tissue and organ and reach therapeutic purpose with the organ that substitutes the human body loss of function and tissue and progressively become a reality.Stem cell causes revolutionary advancement at medical field probably, thereby has immeasurable medical value and cause global extensive concern and research.It is the field that has development and application prospect 21 century most.Regenerative medicine need possess the condition of three necessity: (1) stem cell, promptly have self, propagation and differentiation capability highly, and can repair the cell of impaired organ-tissue; (2) support, the growth of sustenticular cell; (3) growth and differentiation factor promote cell proliferation and differentiation.Wherein stem cell plays a part very importantly, and people find that the earliest embryonic stem cell is can infinite multiplication and to three germinal layers differentiation, but there is ethics problem in embryonic stem cell.
Mescenchymal stem cell (mesenchymal stem cells, be called for short MSCs) be the multipotential stem cell that derives from the mature tissue, having height self ability and multidirectional differentiation potential, is the cenospecies daughter cell of Transplanted cells and organizational project, has broad application prospects.Wherein, research MSCs the most widely is the mescenchymal stem cell that derives from marrow, reduces although its ethics problem is compared with embryonic stem cell greatly, and medullary cell must obtain by the approach of invading, and with advancing age, stem cell population significantly reduces.Many research institutions search out multipotential stem cell in other tissue, the peripheral blood, bleeding of the umbilicus, deciduous teeth, umbilical cord, the amniotic mesenchymal cell that comprise mobilization, yet, the cell that obtains from these tissues is still using foetal calf serum to cultivate in culturing process, there is the practical problems of heteroimmunity originality aspect, limited wide clinical application.
Summary of the invention
The present invention is directed to existing source of human stem cell or be subjected to ethics restriction or a limited number of problem, and allogeneic, wide material sources are provided, turn waste into wealth, be not subjected to a kind of human amnion mesenchymal stem cell serum-free culture medium and the cultural method thereof of ethics restriction and non-immunogenicity problem.
A kind of human amnion mesenchymal stem cell serum-free culture medium of the present invention comprises:
Basic medium DMEM/F12 15.0-15.6g/L
Human serum albumin 5.0-10 * 10
-1%
Human transferrin 1.0-5.5 * 10
-1G/L
Insulin human 5.0-10 * 10
-1G/L
Sodium Selenite 5.2-6.7 * 10
-6G/L.
A kind of human amnion mesenchymal stem cell serum-free cultural method of the present invention may further comprise the steps:
(1) preparation of human amnion mesenchymal stem cell separation and single cell suspension
Get people's amnion 5 * 5cm
2, shred, earlier with final concentration 1.25-2.5g/L trypsinase as far as possible, room temperature digestion 30-60 minute, 2-4 time altogether, end trypsinase with the DMEM/F12 nutrient solution then, employing contains and adds final concentration is the V of 1.0-2.0g/L collagenase IV and 0.1-0.2g/L deoxyribonuclease I
DMEM: V
F12=1: 1 DMEM/F12 nutrient solution, 37 ℃ digested 30-60 minute, and obtained cell suspension; Cell suspension is filtered, make single cell suspension;
(2) human amnion mesenchymal stem cell serum-free cultivation, purifying and amplification
In substratum, placing 37 ℃, saturated humidity, volume fraction is 5% CO with (1) step gained cell inoculation
2, cultivate in the incubator, and, make human amnion mesenchymal stem cell obtain amplification and purifying gradually by changing liquid and going down to posterity;
Described substratum adopts V
DMEM: V
F12=1: 1DMEM/F12 serum free medium 15.0-15.6g/L, wherein contain volume fraction 5.0-10 * 10
-1The % human serum albumin, 1.0-5.5 * 10
-1The g/L human transferrin, 5.0-10 * 10
-1The g/L insulin human, 5.2-6.7 * 10
-6The g/L Sodium Selenite.
In above-mentioned (2) step, select according to qualifications (1) step gained cell with 2 * 10
7L
-1-2 * 10
8L
-1Density is inoculated in the substratum and cultivates.
The process of cell amplification and purifying is preferably as follows: after (2) step beginning cell cultures, cultivated 48-72 hour, change nutrient solution, discard not adherent cell, according to the cell growing state, every 2-3 days full dose is changed liquid once, when treating that cell reaches the 80%-90% fusion, with final concentration 2.5g/L tryptic digestion, then in the ratio of 1: 2 ratio or 1: 3 inoculation culture that goes down to posterity, and being designated as P1 generation, every 2-3 days full dose is changed liquid in the culturing process that goes down to posterity, and merges each other until attached cell, at the bottom of being paved with bottle, repeat the aforesaid operations cultivation of going down to posterity, and be designated as P2 generation, continue the above-mentioned culturing process that goes down to posterity.
The invention has the advantages that: people's amnion is the later waste of fetus birth, the present invention is from the external serum-free culture human amnion mesenchymal stem cell of people's amnion (Human Amniotic mesenchymal stem cells, be called for short HAMSCs), with compare with the nutrient solution cultivator amnion mesenchymal stem cell that contains foetal calf serum, have and do not have that other are animal derived, wide material sources, be not subjected to superiority such as ethics restriction and heteroimmunity originality.
Description of drawings
Fig. 1 (P0-P5) is former HAMSCs inverted microscope (* 40) figure that is commissioned to train foster.
Fig. 2-1 identifies the expression figure of cluster of differentiation among the HAMSCs (cluster of differentiation is called for short CD) 44 for flow cytometry.
Fig. 2-2 identifies the expression figure of CD90 among the HAMSCs for flow cytometry.
Fig. 2-3 identifies the expression figure of CD105 among the HAMSCs for flow cytometry.
Fig. 3-1 is CD44 immunofluorescence dyeing (* 100) figure of HAMSCs cell.
Fig. 3-2 is CD73 immunofluorescence dyeing (* 40) figure of HAMSCs cell.
Fig. 3-3 is CD90 immunofluorescence dyeing (* 100) figure of HAMSCs cell.
Fig. 4-1 is divided into adipocyte (* 100) figure for external evoked HAMSCs.
Fig. 4-2 is divided into scleroblast (* 100) figure for external evoked HAMSCs.
Fig. 4-3 is divided into chondrocyte (* 100) figure for external evoked HAMSCs.
Embodiment
Describe the present invention in detail below in conjunction with drawings and Examples:
A kind of human amnion mesenchymal stem cell serum-free culture medium of the present invention comprises:
Basic medium DMEM/F12 15.6g/L
(mixing in by volume 1: 1 is got 15.6g and is dissolved in the 1000ml water supplier GIBCO)
Human serum albumin 8.0 * 10
-1%
Human transferrin 5.0 * 10
-1G/L
The insulin human 8.0 * 10
-1G/L
Sodium Selenite 6.0 * 10
-6G/L
Be made for the aqueous solution.
Embodiment 2
A kind of human amnion mesenchymal stem cell serum-free cultural method of the present invention may further comprise the steps:
1, the preparation of the separation of human amnion mesenchymal stem cell and single cell suspension
Under aseptic condition, get people's placenta of normal mature c-section fetus (male sex); Detecting other relevant infectivity indexs such as hepatitis A antibody, hepatitis B virus surface antigen, anti-HBs, hepatitis B virus e antigen, antihepatitis b e antibody, hepatitis B core antibody IgM, antibody to hepatitis C, hev antibody, antibody of AIDS virus, syphilis helicoid antibody all is negative; Passivity is separated the amnion 5 * 5cm of placenta umbilical cord face on aseptic super clean bench
2, with fully flushing of PH7.2 phosphoric acid buffer (PBS), amnion is placed the physiological saline that contains 0.1 ten thousand U/ml gentamicins and 2.5ug/ml amphotericin B, soaked 20 minutes; Amnion is shredded as much as possible, add final concentration 2.5g/L trypsinase 5ml, stir and make it fully mixed, insert 37 ℃ of incubator digestion and get the centrifugal 5min of Digestive system 1000rpm after 30 minutes, abandon supernatant liquor with amnion by every gram tissue; Totally 3 times so, use V then
DMEM: V
F12=1: 1DMEM/F12 nutrient solution 5ml ends trypsinase, to remove epithelial cell as far as possible; Add the V that final concentration is 1.0g/L collagenase IV and 0.1g/L deoxyribonuclease I then
DMEM: V
F12=1: 1 DMEM/F12 nutrient solution 5ml places 37 ℃ of digestion to get cell suspension after 30-60 minute; With the filtration of 200 order stainless (steel) wires the cell that digests is made single cell suspension, the centrifugal 5-10 of 1000r/m minute, wash 2 times with PBS, expect blue dyeing with platform, living cell counting is with 2 * 10
8L
-1Density with the gained cell inoculation in the 75ml culturing bottle; Cell density when inoculation can adopt 2 * 10
7-2 * 10
8L
-1
2, the serum-free culture of human amnion mesenchymal stem cell, purifying and amplification
Above-mentioned human amniotic mesenchymal cell is seeded in the 75ml culturing bottle cultivates, include the 5ml serum-free medium, comprising:
Basic medium DMEM/F12 (mixing in by volume 1: 1) 15.0g/L
Human serum albumin 5.0 * 10
-1%
Human transferrin 1.0 * 10
-1G/L
The insulin human 5.0 * 10
-1G/L
Sodium Selenite 5.2 * 10
-6G/L
Cell density during inoculation adopts 2 * 10
7L
-1
Placing 37 ℃ of volume fractions is 5% CO
2Cultivate in the saturated humidity incubator.Cultivate after 48-72 hour, change nutrient solution, discard not adherent cell, according to the cell growing state, every 2-4 days full dose is changed liquid once; When treating that cell reaches 80%-90% and merges, use the 2.5g/L tryptic digestion,, and be designated as P1 generation then in the ratio of the ratio of 1: 2 or 1: 3 inoculation culture that goes down to posterity.Per 2 days full doses are changed liquid in the culturing process that goes down to posterity, and merge each other until attached cell, at the bottom of being paved with bottle, repeat aforesaid operations again and go down to posterity, and this goes down to posterity to cultivate and is designated as P2 generation, continues the above-mentioned culturing process that goes down to posterity then; After passing for 3 generations, cell grows into logarithmic phase, and the doubling time is 2 days.It is fibrous that HAMSCs is that slender type becomes, and is the whirlpool shape after the fusion.Along with the carrying out of going down to posterity, the cell space of HADMSCs increases gradually, the roomy flat cell of part occurs, lose propagation and differentiation capability, and most of cell is still kept elongated fusiformis, keeps hyperplasia and differentiation capability; Vitro culture is after 8 generations, and the rate of propagation of cell obviously slows down, and catabiosis appears in cell.
The separation of a kind of human amnion mesenchymal stem cell of the present invention and serum-free culture method may further comprise the steps:
The 1st step of the present invention can also be: the trypsinase final concentration is 2.5g/L, and digestion time is 30-60 minute, and number of times is 2-4 time; End that the human serum albumin volume fraction can be 5.0 * 10 in the tryptic DMEM/F12 nutrient solution
-1%; When collagenase IV, deoxyribonuclease I digestion, V
DMEM: V
F12The concentration of collagenase IV adopts 0.5-1.5g/L in=1: 1 the DMEM/F12 nutrient solution, deoxyribonuclease 0.05-0.15g/L, and digestion time is 1-2 hour; All the other are with embodiment 1.
Embodiment 4
A kind of human amnion mesenchymal stem cell serum-free cultural method of the present invention comprises the steps:
1, the preparation of the separation of human amnion mesenchymal stem cell and single cell suspension
The amnion fetched is washed bloodstain repeatedly with PBS (the Phosphate buffer saline) phosphate buffered saline buffer of PH7.2, with big tweezers the fine hair rete is peeled off, discard, amnion places microbiotic liquid 50ml to soak 20min; After the PBS flushing, be divided into about 5 * 5cm with scissors
2Place glass dish respectively, shred as far as possible, adding final concentration is 2.5g/L trypsinase 5ml, and 37 ℃ of incubator digestion 30min are transferred in the 15ml centrifuge tube, and the centrifugal 5min of 1000rpm abandons supernatant liquor, digests so altogether 3 times.The DMEM/F12 that adds equivalent during last time, 5ml stops digestion, and is centrifugal, abandons supernatant, and PBS washes repeatedly, and is centrifugal again, abandons supernatant; Adding final concentration is collagenase IV and the 0.1g/L DNA enzyme I mixed solution 5ml of 1.0g/L, places 37 ℃ of incubator digestion 1 hour, adds the DMEM/F12 nutrient solution 5ml of equivalent volumes, and mixing stops digestion; The cell sieve is placed on the diameter 10cm glass dish, will go up step liquid and pour upward filtration collecting cell of cell sieve into, the liquid of collecting is transferred in the 15ml centrifuge tube the centrifugal 5min of 1000rpm, collecting cell;
Abandon supernatant behind the centrifugal with the collection of last step, with DMEM/F12 nutrient solution 5ml re-suspended cell precipitation, with 2 * 10
8/ L is inoculated in 25cm
2In the culturing bottle;
2, the serum-free culture of human amnion mesenchymal stem cell, purifying and amplification
Above-mentioned human amniotic mesenchymal cell is seeded in 25cm
2Culturing bottle is cultivated, and includes the 5ml serum-free medium, and wherein: basic medium is V
DMEM: V
F12=1: 1 DMEM/F12 15.6g/L nutrient solution 5ml comprises volume fraction 1.0 * 10
-1% human serum albumin, 5.5 * 10
-1G/L human transferrin, 10 * 10
-1G/L insulin human and 6.7 * 10
-6The g/L Sodium Selenite, the cell density during inoculation adopts 2 * 10
8L
-1
Placing 37 ℃ of volume fractions is 5% CO
2Cultivate in the saturated humidity incubator, be designated as P
0According to the adherent situation of cell, changed liquid once with DMEM/F12 nutrient solution 5ml full dose in 1-2 days; When treating that cell grows to 80%-90% and merges,, be designated as P in 1: 3 the ratio inoculation culture that goes down to posterity
1By that analogy.
Embodiment 5
The present invention adopts flow cytometry to identify that amnion mesenchymal stem cell is as follows:
1, collecting cell: when treating that cell density reaches 90% fusion, discard the serum free medium nutrient solution, with 1 * PBS5ml rinsing twice of precooling, abandon and add 0.25% pancreatin 1ml behind the clean PBS, observation of cell form under the inverted microscope, behind cell rounding, add 2ml serum free medium nutrient solution and stop digestion, transitional cell centrifugal 5 minutes of 800rpm to the 1.5ml centrifuge tube abandons supernatant, with 1 * PBS 1ml re-suspended cell precipitation of precooling, with same condition repeated centrifugation once, abandon supernatant;
2, anti-hatching: with 500 μ l antibody dilution buffers (1 * PBS, 2% foetal calf serum FBS) dilution in 1: 100 one anti-CD105, CD44,1: 50 dilution one anti-CD73, slow on ice shaking hatched 45 minutes, and centrifugal 5 minutes of 2000rpm abandons supernatant;
3, washing: with 1 * PBS 1ml re-suspended cell precipitation, centrifugal 5 minutes of 2000rpm abandons supernatant.
4, two anti-hatching: with 500 μ l antibody dilution buffer (1 * PBS, 2%FBS) dilute the anti-mouse of two anti-fluorescein isothiocyanate (FITC) link coupled donkeys, the anti-goat of FITC link coupled donkey at 1: 500, slow on ice shaking hatched 30 minutes, and centrifugal 5 minutes of 2000rpm abandons supernatant;
5, washing: with 1 * PBS 1ml re-suspended cell precipitation, centrifugal 5 minutes of 2000rpm abandons supernatant;
6, streaming detects: the Paraformaldehyde 96 300 μ l re-suspended cells precipitation with 1%, in the streaming pipe, detect cell transfer in flow cytometer.
Embodiment 6
The present invention adopts immunofluorescence technique to identify that amnion mesenchymal stem cell is as follows:
1, cell climbing sheet: the clean cover plate that will handle well, insert in six orifice plates, with 2 * 10
5Density is inoculated into HAMSCs and puts into cover plate section six orifice plates, and placing 37 ℃ of volume fractions is 5% CO
2Cultivate in the saturated humidity incubator;
2, fixing: when treating that cell growth 70-80% merges, discard nutrient solution, wash twice with 1 * PBS, add 4% paraformaldehyde solution, room temperature is fixed 20 minutes;
3, washing: the exhaustion Paraformaldehyde 96 adds 1 * PBS, 50rpm/min washing 5 minutes, triplicate;
4, sealing: add confining liquid (containing among the PBS of 1% bSA (BSA)), room temperature sealing 30 minutes;
5, anti-hatching: (CD73 covers the cover plate surface and inserts in the wet box, 4 ℃, spends the night for 1 * PBS, 1%BSA) 1: 100 dilution one anti-CD90, CD44 with 500 μ l antibody dilution buffers;
6, washing: the exhaustion Paraformaldehyde 96 adds 1 * PBS 1ml, 50rpm/min washing 5 minutes, triplicate;
7, two anti-hatching: (1 * PBS 1%BSA) dilutes the anti-mouse of two anti-FITC link coupled donkeys, the anti-goat of tetramethyl rhodaine isothiacyanate (TRITC) link coupled donkey, incubated at room 1 hour at 1: 500 with 500 μ l antibody dilution buffers;
8, washing: the exhaustion Paraformaldehyde 96 adds 1 * PBS 1ml, 50rpm/min washing 5 minutes, triplicate;
9, nuclear staining: with deionized water (ddH
2O) by 1: 1000 dilution proportion two amidine Phenylindole (DAPI) storage liquid, working fluid concentration was 1 μ g/ml, dyeed 1 minute, used ddH
2O gives a baby a bath on the third day after its birth inferior, each 5 minutes;
10, mounting:, place under the fluorescent microscope and observe with 95% glycerine mounting.
Embodiment 7
The present invention induces amnion mesenchymal stem cell to comprise to fat, bone, chondrocyte's differentiation:
One. induce amnion mesenchymal stem cell to break up to adipocyte
1, the HAMSCs kind that obtains is gone in six orifice plates cultivates with serum free medium, treat to be replaced by the DMEM substratum 3ml that inducing culture contains 10%FBS when cell is looked 70% fusion, add 1 micromole (μ M) dexamethasone, 0.5 μ M isobutyl methyl xanthine, 60 μ M indomethacins and 170 μ M Regular Insulin;
2, changed inducing culture 1 time in per 3 days, 3ml cultivated 14 days;
3, after cultivation stops, add 2% Paraformaldehyde 96 room temperature and fix 10 minutes, Virahol 2ml washing with 60%;
4, oil red O incubated at room is 10 minutes, places microscopically to observe.
Two. induce amnion mesenchymal stem cell to osteoblast differentiation
1, the HAMSCs kind that obtains is gone in six orifice plates, treat to be replaced by the DMEM substratum 3ml that contains 10%FBS when cell is looked 70% fusion, add 0.1 μ M dexamethasone, 50 μ g/mL xitix and 10mM glycerophosphate;
2, changed liquid 1 time in per 3 days, 3ml cultivated 21 days;
3, after cultivation stopped, the Paraformaldehyde 96 2ml of adding 4% fixed 10 minutes, used alkaline phosphatase staining (BCIP/NBT) incubated at room 2 hours, and distillation is washed one time, and mirror is detection down; Three. induce amnion mesenchymal stem cell to break up to the chondrocyte
1, with the HAMSCs that obtains with 2 * 10
5Plant in six orifice plates, nutrient solution is replaced by the human serum albumin nutrient solution of serum free medium nutrient solution and 0.5%.And add 50 μ g/ml xitix, 100 μ g/ml Sodium.alpha.-ketopropionates, 0.1 μ M dexamethasone and 10ng/ml transforming growth factor-beta 1 (TGF-β 1);
2, change liquid every other day, now add when TGF-β 1 changes liquid at every turn, cultivated 22 days;
3, after cultivation stopped, the Paraformaldehyde 96 of adding 4% was fixed 10 minutes, and Ai Qianlan (Alcianblue) dyeing is spent the night, and microscopically is observed.
Embodiment 7
Below in conjunction with accompanying drawing result of the present invention is described below:
1, the former foster result that is commissioned to train of amnion mesenchymal stem cell
It is adherent that the HAMSCs that former generation obtains cultivates beginning in 6 hours, and the former foster back of being commissioned to train is adherent rounded, and the HAMSCs that former generation obtains after 4-5 days begins to be stretched to into fibrous.The HAMSCs that two week backs former generations obtain be the colony growth (Fig. 1, P0).The cell colony that forms is gone down to posterity with trysinization, be designated as P1 (Fig. 1, P1).The cell speed of growth after going down to posterity is accelerated gradually, and adherent HAMSCs is the fusiformis stretch-like.After passing for 2,3 generations, cell grows into logarithmic phase, and the doubling time is 2 days.It is fibrous that HAMSCs is that slender type becomes, be after the fusion whirlpool shape (Fig. 1, P3-P5).
Fig. 1, the former foster HAMSCs that is commissioned to train, inverted microscope (* 40) obtains human amnion mesenchymal stem cell (HAMSCs) from the former generation separation of people's amnion, is the cell that obtains in former generation, and P1-P5 is the HAMSCs after going down to posterity.Cell growth is vigorous, and it is fibrous to be elongated one-tenth.
2, amnion mesenchymal stem cell flow cytometry qualification result
CD44 is a kind of cell surface glycoprotein, belongs to the glutinous even molecule family of cell, the interaction in the mediating growth process between cell and the cell.CD90 is also referred to as Thy-1, is a kind of surface marker of stem cell.CD105 is SH2, belongs to Endoglin cell surface molecule.CD44, CD90, CD105 molecule positive expression in mesenchymal stem cells MSCs are a kind of important surface markers of mesenchymal cell.Utilize flow cytometry to identify the same positive expression CD44 of HAMSCs, CD90, CD105 (Fig. 2-1).
3, amnion mesenchymal stem cell identified by immunofluorescence result
Fig. 3-1, the CD44 immunofluorescence dyeing (* 100) of HAMSCs cell, vitro culture HAMSCs, and dye through the cellular immunofluorescence method.That redness is represented is CD44, mainly expresses on film.Blueness is the nucleus of redying with DAPI, observes obtaining image under fluorescent microscope.
Fig. 3-2, the CD73 immunofluorescence dyeing (* 40) of HAMSCs cell, vitro culture HAMSCs, and dye through the cellular immunofluorescence method.That green is represented is CD73, mainly expresses on film.Blueness is the nucleus of redying with DAPI, observes obtaining image under fluorescent microscope.
Fig. 3-3, the CD90 immunofluorescence dyeing (* 100) of HAMSCs cell, vitro culture HAMSCs, and dye through the cellular immunofluorescence method.That redness is represented is CD90, mainly expresses on film.Blueness is the nucleus of redying with DAPI, observes obtaining image under fluorescent microscope.
4, induce amnion mesenchymal stem cell to fat, bone, chondrocyte's differentiated result
Induce amnion mesenchymal stem cell to break up shown in Fig. 4-1 to adipocyte, the mescenchymal stem cell cell is to derive to grow mesoblastic cell, has the potentiality to the mesoblastema differentiation.Vitro culture HAMSCs can induce this cell to scleroblast, fat and chondrocyte's differentiation.Induce HAMSCs can see intracellular little vacuole, it can be dyed redness with oil red 0 dyeing to the fat differentiation.Fig. 4-1, external evoked HAMSCs are divided into adipocyte (* 100), and HAMSCs is induced to differentiate into adipocyte with vitro culture, and oil red O dyes redness with fat granule.
Induce amnion mesenchymal stem cell to osteoblast differentiation shown in Fig. 4-2, scleroblast derives from mesoblastema, contains alkaline phosphatase in the sophisticated scleroblast.Fig. 4-2, external evoked HAMSCs are divided into scleroblast (* 100), and HAMSCs is induced to differentiate into scleroblast with vitro culture, and the alkaline phosphatase staining that detects in the cell cytosol after inducing is a bluish voilet.Induce amnion mesenchymal stem cell to break up shown in Fig. 4-3 to the chondrocyte, the chondrocyte derives from mesoblastema, and the chondrocyte is contained abundant acidic substance and Saliva Orthana, can be dyed blueness by Ai Qian indigo plant.Fig. 4-3, external evoked HAMSCs are divided into chondrocyte (* 100), and HAMSCs is induced to differentiate into the chondrocyte with vitro culture, and the blue dyeing of the cell Ai Qian after inducing is for blue, and Ai Qian indigo plant can be dyed blueness with the acid Saliva Orthana in the cartilage.
Claims (4)
1. human amnion mesenchymal stem cell serum-free culture medium comprises:
Basic medium DMEM/F12 15.0-15.6g/L
Human serum albumin 5.0-10 * 10
-1%
Human transferrin 1.0-5.5 * 10
-1G/L
Insulin human 5.0-10 * 10
-1G/L
Sodium Selenite 5.2-6.7 * 10
-6G/L.
2. human amnion mesenchymal stem cell serum-free cultural method may further comprise the steps:
(1) preparation of human amnion mesenchymal stem cell separation and single cell suspension
Get people's amnion earlier with final concentration 2.5g/L trypsinase, room temperature digestion 30-60 minute 2-4 time altogether, is used V then
DMEM: V
F12=1: the 1DMEM/F12 nutrient solution is ended trypsinase, and employing contains and adds final concentration is the DMEM/F12 nutrient solution of 1.0-2.0g/L collagenase IV and 0.1-0.2g/L deoxyribonuclease I, and 37 ℃ digested 30-60 minute, and obtained cell suspension; Cell suspension is filtered, make single cell suspension;
(2) serum-free culture of human amnion mesenchymal stem cell, purifying and amplification
In substratum, placing 37 ℃, saturated humidity, volume fraction is 5% CO with (1) step gained cell inoculation
2Cultivate in the incubator, and, make human amnion mesenchymal stem cell obtain amplification and purifying gradually by changing liquid and going down to posterity;
Described substratum adopts V
DMEM: V
F12=1: 1DMEM/F12 serum free medium 15.0-15.6g/L, wherein contain volume fraction 5.0-10 * 10
-1The % human serum albumin, 1.0-5.5 * 10
-1The g/L human transferrin, 5.0-10 * 10
-1The g/L insulin human, 5.2-6.7 * 10
-6The g/L Sodium Selenite.
3. a kind of human amnion mesenchymal stem cell serum-free cultural method according to claim 2, further comprising the steps of:
In above-mentioned (2) step, select according to qualifications (1) step gained cell with 2 * 10
7L
-1-2 * 10
8L
-1Density is inoculated in the substratum and cultivates.
4. a kind of human amnion mesenchymal stem cell serum-free cultural method according to claim 2, further comprising the steps of:
The process of cell amplification and purifying is preferably as follows: after (2) step beginning cell cultures, cultivated 48-72 hour, change nutrient solution, discard not adherent cell, according to the cell growing state, full dose was changed liquid once in 2-3 days, when treating that cell reaches the 80%-90% fusion, with final concentration 2.5g/L tryptic digestion, then in the ratio of 1: 2 ratio or 1: 3 inoculation culture that goes down to posterity, and being designated as P1 generation, every 2-3 days full dose is changed liquid in the culturing process that goes down to posterity, and merges each other until attached cell, at the bottom of being paved with bottle, repeat the aforesaid operations cultivation of going down to posterity, and be designated as P2 generation, continue the above-mentioned culturing process that goes down to posterity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010252201A CN101914490B (en) | 2010-08-13 | 2010-08-13 | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010252201A CN101914490B (en) | 2010-08-13 | 2010-08-13 | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101914490A true CN101914490A (en) | 2010-12-15 |
| CN101914490B CN101914490B (en) | 2012-10-10 |
Family
ID=43322136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010252201A Active CN101914490B (en) | 2010-08-13 | 2010-08-13 | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101914490B (en) |
Cited By (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433302A (en) * | 2011-12-15 | 2012-05-02 | 成都清科生物科技有限公司 | Serum-free culture medium for mesenchymal stem cells |
| CN102634482A (en) * | 2012-01-13 | 2012-08-15 | 成都美进生物科技有限公司 | Serum-free complete medium for mesenchymal stem cell |
| CN103160463A (en) * | 2013-04-08 | 2013-06-19 | 哈尔滨体育学院 | Separation method for amniotic mesenchymal stem cells of excellent ice-snow sportsmen |
| CN103243071A (en) * | 2013-05-09 | 2013-08-14 | 陈云燕 | Clinical-grade human mesenchymal stem cell serum-free complete medium |
| CN103422176A (en) * | 2013-07-29 | 2013-12-04 | 庞希宁 | Construction method of human amniotic mesenchymal stem cell bank |
| CN103642751A (en) * | 2013-12-06 | 2014-03-19 | 上海同泽和济生物科技有限公司 | Method for preparing stem cells from human amniotic membrane |
| CN105101965A (en) * | 2013-03-15 | 2015-11-25 | 安皮奥制药股份有限公司 | Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same |
| CN105420185A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for separating and extracting hUC-MSC from umbilical cord outer layer amnion tissue |
| CN105420189A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Serum-free medium and preparing method and application thereof |
| CN105420186A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Kit for cell culture |
| CN105420184A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue |
| CN106282101A (en) * | 2016-11-02 | 2017-01-04 | 遵义医学院附属医院 | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application |
| CN106701673A (en) * | 2017-01-20 | 2017-05-24 | 北京天晟宇生物科技有限公司 | Special culturing medium for human amniotic membrane mesenchymal stem cell |
| CN107653223A (en) * | 2017-11-06 | 2018-02-02 | 江西瑞济生物工程技术股份有限公司 | A kind of amnion stem cell media and its cultural method |
| CN108254237A (en) * | 2017-12-12 | 2018-07-06 | 浙江海洋大学 | A kind of live cell fluorescent colouring method |
| CN109022356A (en) * | 2018-08-30 | 2018-12-18 | 丰泽康生物医药（深圳）有限公司 | A kind of serum free medium improving mesenchymal stem cells into chondrocytes differentiation |
| CN110272859A (en) * | 2019-04-15 | 2019-09-24 | 中国辐射防护研究院 | A kind of cultural method of attached cell |
| CN110331130A (en) * | 2019-07-03 | 2019-10-15 | 依科赛生物科技（太仓）有限公司 | Mesenchymal stem cell serum-free medium and application thereof |
| CN110749579A (en) * | 2019-08-29 | 2020-02-04 | 西安医学院 | Method for detecting influence of drug on pancreatic islet cell lipid uptake through immunofluorescence |
| CN111139221A (en) * | 2020-01-09 | 2020-05-12 | 赛瑞诺(北京)生物科技有限公司 | Culture and cryopreservation method of amniotic mesenchymal stem cells |
| CN112029717A (en) * | 2020-09-17 | 2020-12-04 | 英科博雅基因科技（天津）有限公司 | Serum-free in vitro domestication culture of human mesenchymal stem cells |
| CN113262295A (en) * | 2021-03-09 | 2021-08-17 | 中国医科大学 | Identification and application of human amniotic mesenchymal stem cell exocrine protein POSTN |
| CN115322964A (en) * | 2022-08-18 | 2022-11-11 | 宁波希诺赛生物科技有限公司 | Method for constructing 3D culture amniotic mesenchymal stem cell seed bank |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570090A (en) * | 2004-05-11 | 2005-01-26 | 戴育成 | Cell suspension culture and anchorage culture serum free medium and its preparation method |
| CN101412985A (en) * | 2007-10-15 | 2009-04-22 | 华东理工大学 | Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells |
-
2010
- 2010-08-13 CN CN201010252201A patent/CN101914490B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570090A (en) * | 2004-05-11 | 2005-01-26 | 戴育成 | Cell suspension culture and anchorage culture serum free medium and its preparation method |
| CN101412985A (en) * | 2007-10-15 | 2009-04-22 | 华东理工大学 | Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 《中国优生与遗传杂志》 20061231 于海微等 人脐带血间充质干细胞的研究现状与展望 125-127，130 1-4 第14卷, 第11期 2 * |
Cited By (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433302A (en) * | 2011-12-15 | 2012-05-02 | 成都清科生物科技有限公司 | Serum-free culture medium for mesenchymal stem cells |
| CN102433302B (en) * | 2011-12-15 | 2013-07-03 | 成都清科生物科技有限公司 | Serum-free culture medium for mesenchymal stem cells |
| CN102634482A (en) * | 2012-01-13 | 2012-08-15 | 成都美进生物科技有限公司 | Serum-free complete medium for mesenchymal stem cell |
| CN102634482B (en) * | 2012-01-13 | 2013-08-28 | 成都美进生物科技有限公司 | Serum-free complete medium for mesenchymal stem cell |
| CN105101965B (en) * | 2013-03-15 | 2021-03-09 | 安皮奥制药股份有限公司 | Compositions for stem cell mobilization, homing, expansion and differentiation and methods of use thereof |
| US11026940B2 (en) | 2013-03-15 | 2021-06-08 | Ampio Pharmaceuticals, Inc. | Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same |
| CN105101965A (en) * | 2013-03-15 | 2015-11-25 | 安皮奥制药股份有限公司 | Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same |
| CN103160463B (en) * | 2013-04-08 | 2015-01-14 | 哈尔滨体育学院 | Separation method for amniotic mesenchymal stem cells of excellent ice-snow sportsmen |
| CN103160463A (en) * | 2013-04-08 | 2013-06-19 | 哈尔滨体育学院 | Separation method for amniotic mesenchymal stem cells of excellent ice-snow sportsmen |
| CN103243071A (en) * | 2013-05-09 | 2013-08-14 | 陈云燕 | Clinical-grade human mesenchymal stem cell serum-free complete medium |
| CN103422176B (en) * | 2013-07-29 | 2015-01-07 | 庞希宁 | Construction method of human amniotic mesenchymal stem cell bank |
| CN103422176A (en) * | 2013-07-29 | 2013-12-04 | 庞希宁 | Construction method of human amniotic mesenchymal stem cell bank |
| CN103642751A (en) * | 2013-12-06 | 2014-03-19 | 上海同泽和济生物科技有限公司 | Method for preparing stem cells from human amniotic membrane |
| CN105420184A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue |
| CN105420189A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Serum-free medium and preparing method and application thereof |
| CN105420186A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Kit for cell culture |
| CN105420185A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for separating and extracting hUC-MSC from umbilical cord outer layer amnion tissue |
| CN106282101A (en) * | 2016-11-02 | 2017-01-04 | 遵义医学院附属医院 | A kind of promote the human amnion mesenchymal stem cell method to Chondrocyte Differentiation and application |
| CN106701673A (en) * | 2017-01-20 | 2017-05-24 | 北京天晟宇生物科技有限公司 | Special culturing medium for human amniotic membrane mesenchymal stem cell |
| CN107653223A (en) * | 2017-11-06 | 2018-02-02 | 江西瑞济生物工程技术股份有限公司 | A kind of amnion stem cell media and its cultural method |
| CN108254237A (en) * | 2017-12-12 | 2018-07-06 | 浙江海洋大学 | A kind of live cell fluorescent colouring method |
| CN109022356A (en) * | 2018-08-30 | 2018-12-18 | 丰泽康生物医药（深圳）有限公司 | A kind of serum free medium improving mesenchymal stem cells into chondrocytes differentiation |
| CN110272859A (en) * | 2019-04-15 | 2019-09-24 | 中国辐射防护研究院 | A kind of cultural method of attached cell |
| CN110331130A (en) * | 2019-07-03 | 2019-10-15 | 依科赛生物科技（太仓）有限公司 | Mesenchymal stem cell serum-free medium and application thereof |
| CN110749579A (en) * | 2019-08-29 | 2020-02-04 | 西安医学院 | Method for detecting influence of drug on pancreatic islet cell lipid uptake through immunofluorescence |
| CN111139221A (en) * | 2020-01-09 | 2020-05-12 | 赛瑞诺(北京)生物科技有限公司 | Culture and cryopreservation method of amniotic mesenchymal stem cells |
| CN111139221B (en) * | 2020-01-09 | 2022-05-10 | 赛瑞诺(北京)生物科技有限公司 | Culture and cryopreservation method of amniotic mesenchymal stem cells |
| CN112029717A (en) * | 2020-09-17 | 2020-12-04 | 英科博雅基因科技（天津）有限公司 | Serum-free in vitro domestication culture of human mesenchymal stem cells |
| CN112029717B (en) * | 2020-09-17 | 2023-11-03 | 深圳博雅感知药业有限公司 | Serum-free in vitro domestication culture of human mesenchymal stem cells |
| CN113262295A (en) * | 2021-03-09 | 2021-08-17 | 中国医科大学 | Identification and application of human amniotic mesenchymal stem cell exocrine protein POSTN |
| CN115322964A (en) * | 2022-08-18 | 2022-11-11 | 宁波希诺赛生物科技有限公司 | Method for constructing 3D culture amniotic mesenchymal stem cell seed bank |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101914490B (en) | 2012-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101914490B (en) | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof | |
| CN102002475B (en) | Method for obtaining fat adult stem cells of human and method for establishing stem cell library | |
| CN102127522B (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN104630144B (en) | A kind of separation of umbilical cord blood mesenchymal stem cellses and cultural method | |
| CN102191218B (en) | Complete medium and human amnion-derived mesenchymal stem cell culture method | |
| CN102676451B (en) | Method for separating mesenchymal stem cells from placenta | |
| CN109234229B (en) | Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same | |
| CN107022521A (en) | Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell | |
| CN102676452A (en) | Culture medium containing human umbilical cord mesenchymal stem cell exudates and preparation method and applications thereof | |
| CN104164403A (en) | Method for extracting and culturing adipose-derived stem cells | |
| CN106801032B (en) | Construction method of human amniotic epithelial stem cell bank | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN102533659A (en) | Method for inducing human umbilical cord mesenchymal stem cells into testicular interstitial cells and application thereof | |
| CN107236704A (en) | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used | |
| CN102965335A (en) | Kit for mesenchymal stem cell culture and application thereof | |
| CN102485885A (en) | Separating method and application of fat stem cells | |
| CN105087474A (en) | Culture method of deciduous tooth pulp stem cells | |
| CN1912109A (en) | Structural method and application of tissue engineering adipose tissue | |
| CN104762257A (en) | Method for preparing mesenchymal stem cell from umbilical cord | |
| CN106282108A (en) | A kind of spleen mescenchymal stem cell with immunoloregulation function and preparation method and application | |
| CN102533643A (en) | Method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells | |
| CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
| CN101705209A (en) | Method for separating heart stem cells from brown fat and splitting cardioblast | |
| CN101372682A (en) | Construction method of Epinephelus fuscoguttatus fin cell line |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20181214 Address after: 110000 No. 400-4 Zhihui Second Street, Hunnan District, Shenyang City, Liaoning Province Patentee after: Shenyang Amy bio engineering technology R & D Center Co., Ltd. Address before: 110001, No. two, 92 North Road, Heping District, Liaoning, Shenyang Patentee before: China Medical Univ. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210831 Address after: 110000 floors 1-8, No. 155-5, Chuangxin Road, Shenyang pilot Free Trade Zone, Shenyang, Liaoning Province Patentee after: Liaoning amiao stem cell and Regenerative Medicine Research Institute Co.,Ltd. Address before: 110000 No. 400-4 Zhihui Second Street, Hunnan District, Shenyang City, Liaoning Province Patentee before: SHENYANG AIMEO BIOENGINEERING TECHNOLOGY R&D CENTER Co.,Ltd. |
|
| TR01 | Transfer of patent right |