CN108103015A - A kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell - Google Patents
A kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell Download PDFInfo
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- CN108103015A CN108103015A CN201810115674.4A CN201810115674A CN108103015A CN 108103015 A CN108103015 A CN 108103015A CN 201810115674 A CN201810115674 A CN 201810115674A CN 108103015 A CN108103015 A CN 108103015A
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- 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
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- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell, including step in detail below：Step 1：Umbilical cord processing：After umbilical cord is rinsed 3~5 times with D Hank ' s liquid about 0.7~1.0mm is shredded into scissors3Fritter；Step 2：Digestion：Umbilical cord block obtained in step 1 is added in 0.1% clostridiopetidase A II to digest 30~35 minutes, 0.25% pancreatin is then added and carries out digestion 30~35 minutes.The present invention is by choosing umbilical cord caused by newborn, umbilical cord can be turned waste into wealth, umbilical cord mesenchymal stem cells virus infection chance is small simultaneously, it has no adverse effects to donor and patient, and mescenchymal stem cell competence for added value is strong, good basis is provided to establish mesenchyma stem cell, external condition and time easily monitoring and control in each step simultaneously, poor environment is avoided to cultivating and amplification impacts, so as to ensure that the growth course of mescenchymal stem cell from ectocine, culture and the rate of amplification of mescenchymal stem cell are improved with this.
Description
Technical field
The present invention relates to cell culture and amplification technique field, filled between more particularly to a kind of amplification in vitro and pilot scale culture
The method of matter stem cell.
Background technology
Mescenchymal stem cell is a kind of multipotential stem cell, because it is in hematopoietic stem cell transplantation, graft versus host disease(GVH disease) etc.
Play an important roll, become the hot spot of stem-cell research at present.The main source of mescenchymal stem cell is Adult Human Bone Marrow at present, but
Adult Human Bone Marrow source mescenchymal stem cell cell quantity and Proliferation, Differentiation potential decline with the increase at age, viral infection rate compared with
Height, and the acquisition palpus row bone marrow puncture of donor mescenchymal stem cell, source is restricted, so that mescenchymal stem cell faces
Bed application is restricted.Therefore, a kind of method for inventing amplification in vitro and pilot scale culture mescenchymal stem cell is above-mentioned to solve
Problem is necessary.
The content of the invention
It is an object of the invention to provide a kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell, to solve
The problems mentioned above in the background art.
To achieve the above object, the present invention provides following technical solution：A kind of amplification in vitro and pilot scale culture mesenchyma
The method of stem cell, including step in detail below：
Step 1：Umbilical cord processing：About 0.7 is shredded into scissors after umbilical cord is rinsed 3~5 times with D-Hank ' s liquid~
1.0mm3Fritter；
Step 2：Digestion：Umbilical cord block obtained in step 1 is added in 0.1% clostridiopetidase A II to digest 30~35 minutes,
Then add 0.25% pancreatin and carry out digestion 30~35 minutes；
Step 3：Centrifugation：Solution in step 2 is poured into centrifuge, centrifuge 20~25 minutes, Ran Houqu
Intermediate tunica albuginea layer, and add in PBS and wash 2 times；
Step 4：Culture：Cell inoculation after step 3 is washed is in 20%~40% hyclone culture solution I, so
It is placed on 37 DEG C, cultivated in the incubator of 5% carbon dioxide and saturated humidity, stand 2~3 days；
Step 5：Cultivate fluid exchange：Culture solution in culture solution I after being stood 2~3 days in step 4 is replaced,
And non-adherent cell is removed, while was changed the liquid once every 2~3 days；
Step 6：Digestion：After step 5 progress, culture solution is positioned over micro- Microscopic observation, waits to paste when observing
After parietal cell 80%~90%, the digestion of 0.25% pancreatin is added in；
Step 7：Secondary culture：Suitable cell is drawn from the postdigestive solution of step 6 with suction pipe, and with 2*
104/ ml density is inoculated in 20%~40% hyclone culture solution II and carries out secondary culture, is placed in 37 DEG C, 5% carbon dioxide
It is cultivated in the incubator of saturated humidity, stands 2~3 days, then carry out step 5 and step 6, and so on.
Preferably, umbilical cord is umbilical cord caused by the newborn being just born in step 1, and scissors passes through 300 DEG C~500
The bacterium on DEG C high-temperature sterilization removal scissors surface.
Preferably, it is aseptically to carry out in step 2.
Preferably, centrifuge carries out centrifugal movement with 2000r/min in step 3.
Preferably, 100kU/L penicillin and 100mg/L chains are also contained in step 4 and step 7 in hyclone culture solution
Mycin.
Preferably, the non-adherent cell of the processing in step 5 carries out centralized collection by collecting box.
The technique effect and advantage of the present invention：
1st, the present invention can be turned waste into wealth umbilical cord by choosing umbilical cord caused by newborn, while umbilical cord mesenchyma
Stem cell virus infection chance is small, has no adverse effects to donor and patient, and mescenchymal stem cell competence for added value is strong, to establish
Mesenchyma stem cell provides good basis；
2nd, the required instrument of the present invention and treatment fluid are all existing easily to obtain, and at low cost, so as to drop significantly
Low cost of manufacture；
3rd, external condition and time easily monitoring and control in each step of the present invention, avoid poor environment to culture and
Amplification impacts, so as to ensure that the growth course of mescenchymal stem cell from ectocine, improves mesenchyma with this and does carefully
The culture of born of the same parents and rate of amplification.
Description of the drawings
Fig. 1 is the flow diagram of the present invention.
Specific embodiment
Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work
Embodiment belongs to the scope of protection of the invention.
Embodiment 1：The present invention provides:A kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell, including
Step in detail below：
Step 1：Umbilical cord processing：Umbilical cord D-Hank ' s liquid caused by the newborn being just born is rinsed 3~5 times
Afterwards about 0.7~1.0mm is shredded into the scissors after 300 DEG C~500 DEG C high-temperature sterilizations3Fritter；
Step 2：Digestion：It aseptically carries out, umbilical cord block obtained in step 1 is added in into 0.1% clostridiopetidase A
II digestion 30~35 minutes, then adds 0.25% pancreatin and carries out digestion 30~35 minutes；
Step 3：Centrifugation：Solution in step 2 is poured into centrifuge, centrifuge carries out centrifugation fortune with 2000r/min
It is 20~25 minutes dynamic, intermediate tunica albuginea layer is then taken, and adds in PBS and washs 2 times；
Step 4：Culture：Cell inoculation after step 3 is washed in 20% hyclone, 100kU/L penicillin and
In 100mg/L streptomysins culture solution I, 37 DEG C are subsequently placed in, cultivated in the incubator of 5% carbon dioxide and saturated humidity,
Stand 2~3 days；
Step 5：Cultivate fluid exchange：Culture solution in culture solution I after being stood 2~3 days in step 4 is replaced,
And non-adherent cell is removed, and the non-adherent cell of processing is subjected to centralized collection by collecting box, while every 2~3
It is changed the liquid once；
Step 6：Digestion：After step 5 progress, culture solution is positioned over micro- Microscopic observation, waits to paste when observing
After parietal cell 80%~90%, the digestion of 0.25% pancreatin is added in；
Step 7：Secondary culture：Suitable cell is drawn from the postdigestive solution of step 6 with suction pipe, and with 2*
104/ ml density is inoculated in 20% hyclone, 100kU/L penicillin and 100mg/L streptomysins culture solution II and carries out passage training
It supports, is placed in 37 DEG C, is cultivated in the incubator of 5% carbon dioxide and saturated humidity, stand 2~3 days, then carry out step 5
And step 6, and so on.
Embodiment 2：The present invention provides:A kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell, including
Step in detail below：
Step 1：Umbilical cord processing：Umbilical cord D-Hank ' s liquid caused by the newborn being just born is rinsed 3~5 times
Afterwards about 0.7~1.0mm is shredded into the scissors after 300 DEG C~500 DEG C high-temperature sterilizations3Fritter；
Step 2：Digestion：It aseptically carries out, umbilical cord block obtained in step 1 is added in into 0.1% clostridiopetidase A
II digestion 30~35 minutes, then adds 0.25% pancreatin and carries out digestion 30~35 minutes；
Step 3：Centrifugation：Solution in step 2 is poured into centrifuge, centrifuge carries out centrifugation fortune with 2000r/min
It is 20~25 minutes dynamic, intermediate tunica albuginea layer is then taken, and adds in PBS and washs 2 times；
Step 4：Culture：Cell inoculation after step 3 is washed in 30% hyclone, 100kU/L penicillin and
In 100mg/L streptomysins culture solution I, 37 DEG C are subsequently placed in, cultivated in the incubator of 5% carbon dioxide and saturated humidity,
Stand 2~3 days；
Step 5：Cultivate fluid exchange：Culture solution in culture solution I after being stood 2~3 days in step 4 is replaced,
And non-adherent cell is removed, and the non-adherent cell of processing is subjected to centralized collection by collecting box, while every 2~3
It is changed the liquid once；
Step 6：Digestion：After step 5 progress, culture solution is positioned over micro- Microscopic observation, waits to paste when observing
After parietal cell 80%~90%, the digestion of 0.25% pancreatin is added in；
Step 7：Secondary culture：Suitable cell is drawn from the postdigestive solution of step 6 with suction pipe, and with 2*
104/ ml density is inoculated in 30% hyclone, 100kU/L penicillin and 100mg/L streptomysins culture solution II and carries out passage training
It supports, is placed in 37 DEG C, is cultivated in the incubator of 5% carbon dioxide and saturated humidity, stand 2~3 days, then carry out step 5
And step 6, and so on.
Embodiment 3：The present invention provides:A kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell, including
Step in detail below：
Step 1：Umbilical cord processing：Umbilical cord D-Hank ' s liquid caused by the newborn being just born is rinsed 3~5 times
Afterwards about 0.7~1.0mm is shredded into the scissors after 300 DEG C~500 DEG C high-temperature sterilizations3Fritter；
Step 2：Digestion：It aseptically carries out, umbilical cord block obtained in step 1 is added in into 0.1% clostridiopetidase A
II digestion 30~35 minutes, then adds 0.25% pancreatin and carries out digestion 30~35 minutes；
Step 3：Centrifugation：Solution in step 2 is poured into centrifuge, centrifuge carries out centrifugation fortune with 2000r/min
It is 20~25 minutes dynamic, intermediate tunica albuginea layer is then taken, and adds in PBS and washs 2 times；
Step 4：Culture：Cell inoculation after step 3 is washed in 40% hyclone, 100kU/L penicillin and
In 100mg/L streptomysins culture solution I, 37 DEG C are subsequently placed in, cultivated in the incubator of 5% carbon dioxide and saturated humidity,
Stand 2~3 days；
Step 5：Cultivate fluid exchange：Culture solution in culture solution I after being stood 2~3 days in step 4 is replaced,
And non-adherent cell is removed, and the non-adherent cell of processing is subjected to centralized collection by collecting box, while every 2~3
It is changed the liquid once；
Step 6：Digestion：After step 5 progress, culture solution is positioned over micro- Microscopic observation, waits to paste when observing
After parietal cell 80%~90%, the digestion of 0.25% pancreatin is added in；
Step 7：Secondary culture：Suitable cell is drawn from the postdigestive solution of step 6 with suction pipe, and with 2*
104/ ml density is inoculated in 40% hyclone, 100kU/L penicillin and 100mg/L streptomysins culture solution II and carries out passage training
It supports, is placed in 37 DEG C, is cultivated in the incubator of 5% carbon dioxide and saturated humidity, stand 2~3 days, then carry out step 5
And step 6, and so on.
Pass through Examples 1 to 3, when hyclone content is 30%, the growth of mesenchymal stem cells in culture solution
It is best with expanding effect.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, still may be used
To modify to the technical solution recorded in foregoing embodiments or carry out equivalent substitution to which part technical characteristic,
Within the spirit and principles of the invention, any modifications, equivalent replacements and improvements are made should be included in the present invention's
Within protection domain.
Claims (6)
1. a kind of method of amplification in vitro and pilot scale culture mescenchymal stem cell, which is characterized in that including step in detail below：
Step 1：Umbilical cord processing：After umbilical cord is rinsed 3~5 times with D-Hank ' s liquid about 0.7~1.0mm is shredded into scissors3's
Fritter；
Step 2：Digestion：Umbilical cord block obtained in step 1 is added in into 0.1% clostridiopetidase A II and digests 30~35 minutes, then
It adds 0.25% pancreatin and carries out digestion 30~35 minutes；
Step 3：Centrifugation：Solution in step 2 is poured into centrifuge, then centrifuge 20~25 minutes takes centre
Tunica albuginea layer, and add in PBS and wash 2 times；
Step 4：Culture：Then cell inoculation after step 3 is washed is put in 20%~40% hyclone culture solution I
It is cultivated in 37 DEG C, the incubator of 5% carbon dioxide and saturated humidity, stands 2~3 days；
Step 5：Cultivate fluid exchange：Culture solution in culture solution I after being stood 2~3 days in step 4 is replaced, and
Non-adherent cell is removed, while was changed the liquid once every 2~3 days；
Step 6：Digestion：After step 5 progress, culture solution is positioned over micro- Microscopic observation, when observe treat it is adherent thin
After born of the same parents 80%~90%, the digestion of 0.25% pancreatin is added in；
Step 7：Secondary culture：Suitable cell is drawn from the postdigestive solution of step 6 with suction pipe, and with 2*104/ml
Density is inoculated in 20%~40% hyclone culture solution II and carries out secondary culture, is placed in 37 DEG C, 5% carbon dioxide and saturation
It is cultivated in the incubator of humidity, stands 2~3 days, then carry out step 5 and step 6, and so on.
2. the method for a kind of amplification in vitro according to claim 1 and pilot scale culture mescenchymal stem cell, feature exist
In：Umbilical cord is umbilical cord caused by the newborn being just born in step 1, and scissors is gone by 300 DEG C~500 DEG C high-temperature sterilizations
Except the bacterium on scissors surface.
3. the method for a kind of amplification in vitro according to claim 1 and pilot scale culture mescenchymal stem cell, feature exist
In：It is aseptically to carry out in step 2.
4. the method for a kind of amplification in vitro according to claim 1 and pilot scale culture mescenchymal stem cell, feature exist
In：Centrifuge carries out centrifugal movement with 2000r/min in step 3.
5. the method for a kind of amplification in vitro according to claim 1 and pilot scale culture mescenchymal stem cell, feature exist
In：Also contain 100kU/L penicillin and 100mg/L streptomysins in step 4 and step 7 in hyclone culture solution.
6. the method for a kind of amplification in vitro according to claim 1 and pilot scale culture mescenchymal stem cell, feature exist
In：The non-adherent cell of processing in step 5 carries out centralized collection by collecting box.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112159795A (en) * | 2020-10-13 | 2021-01-01 | 肖利霞 | Passage amplification method for umbilical cord mesenchymal stem cell working cells in public bank |
| CN112424335A (en) * | 2018-09-19 | 2021-02-26 | 麦迪康公司 | Self-culture method and device of autologous stem cells |
| CN113621568A (en) * | 2021-07-22 | 2021-11-09 | 江苏蒙彼利生物科技有限公司 | Umbilical cord mesenchymal stem cell serum-free culture equipment and culture method thereof |
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| CN112424335A (en) * | 2018-09-19 | 2021-02-26 | 麦迪康公司 | Self-culture method and device of autologous stem cells |
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| CN113621568A (en) * | 2021-07-22 | 2021-11-09 | 江苏蒙彼利生物科技有限公司 | Umbilical cord mesenchymal stem cell serum-free culture equipment and culture method thereof |
| CN113621568B (en) * | 2021-07-22 | 2024-02-13 | 江苏蒙彼利生物科技有限公司 | Umbilical cord mesenchymal stem cell serum-free culture equipment and culture method thereof |
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