CN106978389A - A kind of combination culture medium for cultivating amniotic epithelial cells - Google Patents
A kind of combination culture medium for cultivating amniotic epithelial cells Download PDFInfo
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- CN106978389A CN106978389A CN201710312034.8A CN201710312034A CN106978389A CN 106978389 A CN106978389 A CN 106978389A CN 201710312034 A CN201710312034 A CN 201710312034A CN 106978389 A CN106978389 A CN 106978389A
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- amniotic
- culture medium
- epithelial cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention discloses a kind of combination culture medium for cultivating amniotic epithelial cells, belongs to field of cell culture.The combination culture medium includes following component：EPLIFE culture medium+1%HKGS+ amniotic homogenate supernatants；Concentration of the albumen in combination culture medium in described amniotic homogenate supernatant is 2~8ng/mL.The combination culture medium of the present invention is will to digest the amnion for removing cell by freeze-drying, liquid nitrogen grinding, ultrasonication, and filtration sterilization obtains amniotic homogenate supernatant, and the homogenate supernatant is added in defined medium with certain proportion.The culture medium can effectively improve amniotic epithelial cells multiplication capacity while amniotic epithelial cells characteristic is maintained, and extend effective culture algebraically of amniotic epithelial cells.
Description
Technical field
The invention belongs to field of cell culture, more particularly to a kind of combination culture medium for cultivating amniotic epithelial cells.
Background technology
Amnion is located at the chorial surface of fetus, is smooth, without blood vessel, impassivity, the transparent membrane without lymph, thick about
0.02~0.50mm, is made up of amniotic epithelial cells, basilar memebrane and matrix.It is formed at the fertilization the 8th day before primitive gut, amnion
The plasticity of primitive gut embryonic cell before histocyte is maintained, the main origin of amnion tissue comes from ectodermic amnioic epithelium
(amniotic epithelial cells, AECs) and from mesoblastic amnion mesenchymal (amniotic
Mesenchyme cells, AMCs) two class cells composition, the differentiation that amniotic epithelial cells have three kinds of germ confluent monolayer cells dives
Energy.
Nerve to occur early stage is thought in biology of nerve growth research, and amnion tissue is directly contacted with neural epithelium, to amniotic fluid
Middle release neurotransmitters and neurotrophic factor, play an important role in nervous system development process.Because amnion tissue is
From the product of fetus, under mother's immune system monitoring, AECs surfaces HLA DR genetic locuses
(human leucocyte antigen-DR, HLA-DR) low expression, does not express HLA-A, B, C antigens, in addition, amnion tissue system
Postpartum, discarded object was without ethnics Problem；Amnion tissue cell derived is abundant, Neurobiology feature and work(with nerve cell
Can, amnion tissue cell is expected to the reliable cell as regenerative medicine field using above-mentioned advantage and originate, carry out cell transplantation
Treat nervous system degenerative disease and traumatic nerve damage.
But it is due to that amniotic epithelial cells are difficult to maintain its stem cell properties in incubation, on the one hand easily breaks up shape
Into ripe epithelial cell, its stem cell properties is lost；On the other hand epithelium-mesenchymal transformation is easily produced, its epithelial cell is lost
Characteristic.It is dfficult to apply in regenerative medicine.
Amniotic epithelial cells will be applied to regenerative medicine, it is necessary first to which the problem of solving is how to be held on chrotoplast
Its multiplication capacity is maintained while characteristic.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, amniotic epithelial cells are cultivated it is an object of the invention to provide one kind
Combination culture medium.
The purpose of the present invention is achieved through the following technical solutions：
A kind of combination culture medium for cultivating amniotic epithelial cells, including following component：
EPLIFE culture medium+1%HKGS+ amniotic homogenate supernatants；
Concentration of the albumen in combination culture medium in described amniotic homogenate supernatant is 2~8ng/mL, preferably 4ng/
mL。
The preparation method of described amniotic homogenate supernatant, comprises the following steps：
(1) amnion is digested by 0.25% pancreatin, and the amniotic epithelial cells adhered to thereon, sterilized water for injection are removed completely
Thoroughly cleaning；
(2) cleaned amnion is shredded, moisture is freezed completely；
(3) liquid nitrogen and lyophilized amnion are poured into mortar, grinding is until liquid nitrogen volatilization repeatedly；
(4) a small amount of basal medium DMEM/F12 is added, is mixed；
(5) ultrasonication, is circulated 30 times；
(6) 0.22 μm of membrane filtrations, take filtrate, as amniotic homogenate supernatant.
The time of digestion described in step (1) is preferably 2h.
The condition of ultrasonication described in step (1) is Ф=6, ultrasonic time 3s, interval time 3s.
The present invention has the following advantages and effect relative to prior art：
The combination culture medium of the present invention is will to digest the amnion for removing cell by freeze-drying, liquid nitrogen grinding, ultrasonication,
Filtration sterilization obtains amniotic homogenate supernatant, and the homogenate supernatant is added in defined medium with certain proportion.The culture medium can be with
Amniotic epithelial cells multiplication capacity is effectively improved while amniotic epithelial cells characteristic is maintained, extends having for amniotic epithelial cells
Effect culture algebraically.
Brief description of the drawings
Fig. 1 be in embodiment 1 P1 for amniotic epithelial cells form.
Fig. 2 be in embodiment 1 P1 for amnioic epithelium CD29 streaming Phenotypic examinations.
Fig. 3 be in embodiment 1 P1 for CK19 SABCs.
Fig. 4 be in embodiment 1 P6 for amniotic epithelial cells form.
Fig. 5 be in embodiment 1 P6 for CK19 SABCs.
Fig. 6 is Test P8 cellular morphologies and SABC in embodiment 1；Wherein, A：Test P8 cellular morphologies；B：Test
P8 SABCs.
Fig. 7 is P6 two groups of amniotic epithelial cells forms of generation in embodiment 2.
Fig. 8 is P6 two groups of amniotic epithelial cells CK19 SABCs of generation in embodiment 2.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1
First, materials and methods
1. material and instrument
(1) material：Amnion sample is derived from the pregnant baby of healthy puerpera.Hepatitis B, hepatitis C, syphilis, AIDS, huge after testing
Cell virus, TORCH detections, mycoplasma, Chlamydia, G-6PD and ground are poor etc. negative.Transport blood bank process guarantor after collection of specimens back
Hold 4~8 DEG C of traffic conditions.
(2) instrument and equipment
Incubator：SANYO GS, model MCO-20AIC
Super-clean bench：The safe and sound air technique Co., Ltds of Su Jing, model SW-CJ-2FD.
Microscope：Olympus, model C KX41-F32FL.
Centrifuge：Thermo, model HeraeusMultifuge X3.
(3) consumptive material：
2~8 DEG C of EPLIFE basal medium gibico companies
- 20 DEG C of HKGS additive gibico companies
2~8 DEG C of pancreatin gibico companies.
2. the preparation of amniotic epithelial cells
Extract amniotic epithelial cells from the amnion tissue of Healthy People, the amniotic epithelial cells of acquisition carry out in vitro culture,
Amplification.
1) the sterile placenta for taking Healthy People, amnion is separated with placental villus tissues, sterile saline cleaning amnion；
2) 37 DEG C of 0.01% pancreatin digests 10 minutes, discards digest, sterile saline cleaning amnion；
3) 37 DEG C of 0.1% pancreatin digests 10 minutes, collects digest and terminates digestion, sterile saline cleaning with serum
Amnion, collects washing lotion, mixture slaking thing and washing lotion.
4) 800g, 10min are centrifuged, and collect cell.
3. it is prepared by amniotic homogenate supernatant
(1) amnion is digested 2 hours by 0.25% pancreatin, and the amniotic epithelial cells adhered to thereon, sterilizing note are removed completely
Penetrate and use water thoroughly cleaning.
(2) cleaned amnion is shredded, freezes moisture in freeze drier completely.
(3) liquid nitrogen and lyophilized amnion are poured into mortar, grinding is until liquid nitrogen volatilization repeatedly.
(4) a small amount of basal medium DMEM/F12 is added, is mixed.
(5) ultrasonication, Ф=6, ultrasonic time 3s, interval time 3s are circulated 30 times.
(6) 0.22 μm of membrane filtrations.
(7) protein concentration is detected, a small amount of basal medium EPLIFE culture mediums is added and adjusts protein concentration to 40ng/mL, 4
DEG C preserve.
4. cell culture
Cell is equally divided into two groups of Test and Control, Test add culture medium EPLIFE+1% (v/v) HKGS+ amnions
It is homogenized the final concentration of 4ng/mL of amniotic homogenate supernatant protein in supernatant, culture medium；Control is EPLIFE+1% (v/v) HKGS,
Cell density is adjusted to 1 × 104/ mL, 2mL/T25 blake bottle culture.
5. the identification of amniotic epithelial cells
The identification of amniotic epithelial cells is by the following method：Amniotic epithelial cells morphological analysis, CK19 SABCs,
CD19 streaming phenotypic evaluations.
2nd, result and analysis：
P1 is observed under inverted microscope more single for amniotic epithelial cells form, the growth of paving stone sample is (see Fig. 1).Through streaming
Cell instrument detection display, cell surface antigen CD29, positive expression rate reach more than 95% (see Fig. 2), and CK19 SABCs show
Show kytoplasm and after birth stained positive (see Fig. 3), it was demonstrated that the cell obtained is amniotic epithelial cells.
6. Secondary Culture
Cell is equally divided into two groups of Test and Control, Test add culture medium EPLIFE+1% (v/v) HKGS+ amnions
It is homogenized the final concentration of 4ng/mL of amniotic homogenate supernatant protein in supernatant, culture medium；Control is EPLIFE+1% (v/v) HKGS,
Cell density is adjusted to 1 × 104/ mL, 2mL/T25 blake bottle culture.Cell 80% is passed on after merging.
7. Secondary Culture result
Control groups P6 is serious for cell senescence, P7 for when cell can not continue adherent growth (see Fig. 4).
Test groups P8 for cell senescence, P9 for when cell can not continue adherent growth (see Fig. 5).
It can be seen that Test groups maintain longer time multiplication capacity compared with control groups, and during breeding, maintain its epithelium thin
Born of the same parents' characteristic (Fig. 6).
Embodiment 2
Amnion sample is derived from the pregnant baby of healthy puerpera.Hepatitis B, hepatitis C, syphilis, AIDS, giant cell are sick after testing
Poison, TORCH detections, mycoplasma, Chlamydia, G-6PD and ground are poor etc. negative.Transport blood bank's process after collection of specimens back and keep 4~8
DEG C traffic condition.Handle in accordance with the following methods.
1. the preparation of amniotic epithelial cells
Extract amniotic epithelial cells from the amnion tissue of Healthy People, the amniotic epithelial cells of acquisition carry out in vitro culture,
Amplification.
1) the sterile placenta for taking Healthy People, amnion is separated with placental villus tissues, sterile saline cleaning amnion；
2) 37 DEG C of 0.01% pancreatin digests 10 minutes, discards digest, sterile saline cleaning amnion；
3) 37 DEG C of 0.1% pancreatin digests 10 minutes, collects digest and terminates digestion, sterile saline cleaning with serum
Amnion, collects washing lotion, mixture slaking thing and washing lotion.
4) 800g, 10min are centrifuged, and collect cell.
2. it is prepared by amniotic homogenate supernatant
(1) amnion is digested 2 hours by 0.25% pancreatin, and the amniotic epithelial cells adhered to thereon, sterilizing note are removed completely
Penetrate and use water thoroughly cleaning.
(2) cleaned amnion is shredded, freezes moisture in freeze drier completely.
(3) liquid nitrogen and lyophilized amnion are poured into mortar, grinding is until liquid nitrogen volatilization repeatedly.
(4) a small amount of basal medium DMEM/F12 is added, is mixed.
(5) ultrasonication, Ф=6, ultrasonic time 3s, interval time 3s are circulated 30 times.
(6) 0.22 μm of membrane filtrations.
(7) protein concentration is detected, a small amount of basal medium EPLIFE culture mediums is added and adjusts protein concentration to 40ng/mL.
P2 is for cellular identification be the same as Example 1
3. cell is equally divided into two groups of Test and Control, Test add culture medium EPLIFE+1% (v/v) HKGS+ sheep
Film is homogenized the final concentration of 4ng/mL of amniotic homogenate supernatant protein in supernatant, culture medium；Culture medium DMEM+ based on Control
10% (v/v) hyclone, adjustment cell density to 1 × 104/ mL, 2mL/T25 blake bottle culture.
4. the identification of amniotic epithelial cells
1) identification of amniotic epithelial cells is by the following method
P6 is identified for rear CK19 SABCs.
2) cellular identification result：
Control shows obvious mesenchyma sample form, and cell is elongated (see Fig. 7), and SABC CK19 dyeing is feminine gender
(see Fig. 8).
Test paving stones sample grows, and cell is small and in polygon (see Fig. 7), and SABC CK19 dyeing is the positive (see figure
8)。
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (5)
1. a kind of combination culture medium for cultivating amniotic epithelial cells, it is characterised in that including following component：
EPLIFE culture medium+1%HKGS+ amniotic homogenate supernatants；
Concentration of the albumen in combination culture medium in described amniotic homogenate supernatant is 2~8ng/mL.
2. the combination culture medium of culture amniotic epithelial cells according to claim 1, it is characterised in that：
Concentration of the albumen in combination culture medium in described amniotic homogenate supernatant is 4ng/mL.
3. the combination culture medium of culture amniotic epithelial cells according to claim 1 or 2, it is characterised in that：
The preparation method of described amniotic homogenate supernatant, comprises the following steps：
(1) amnion digests by 0.25% pancreatin, the amniotic epithelial cells adhered to thereon is removed completely, sterilized water for injection is thorough
Cleaning；
(2) cleaned amnion is shredded, moisture is freezed completely；
(3) liquid nitrogen and lyophilized amnion are poured into mortar, grinding is until liquid nitrogen volatilization repeatedly；
(4) a small amount of basal medium DMEM/F12 is added, is mixed；
(5) ultrasonication, is circulated 30 times；
(6) 0.22 μm of membrane filtrations, take filtrate, as amniotic homogenate supernatant.
4. the combination culture medium of culture amniotic epithelial cells according to claim 3, it is characterised in that：
The time of digestion described in step (1) is 2h.
5. the combination culture medium of culture amniotic epithelial cells according to claim 3, it is characterised in that：
The condition of ultrasonication described in step (1) is Ф=6, ultrasonic time 3s, interval time 3s.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020157195A1 (en) | 2019-01-30 | 2020-08-06 | Univerza V Ljubljani | Procedure for the preparation of an amniotic membrane homogenate based antimicrobial agent |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712020A (en) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | Production of amniotic extractive liquid and use thereof |
| CN101760447A (en) * | 2009-10-30 | 2010-06-30 | 中国人民解放军总医院 | Method for inducing and acclimating epidermal stem cells into nerve cells |
| EP2566583A2 (en) * | 2010-05-06 | 2013-03-13 | Regenics AS | Use of cellular extracts for skin rejuvenation |
| CN104224842A (en) * | 2014-09-03 | 2014-12-24 | 苗九昌 | Preparation method of compound amniotic membrane powder and compound amniotic membrane powder prepared thereby |
| CN104371970A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Culture medium for culturing human amniotic epithelial cells |
-
2017
- 2017-05-05 CN CN201710312034.8A patent/CN106978389A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712020A (en) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | Production of amniotic extractive liquid and use thereof |
| CN101760447A (en) * | 2009-10-30 | 2010-06-30 | 中国人民解放军总医院 | Method for inducing and acclimating epidermal stem cells into nerve cells |
| EP2566583A2 (en) * | 2010-05-06 | 2013-03-13 | Regenics AS | Use of cellular extracts for skin rejuvenation |
| CN104371970A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Culture medium for culturing human amniotic epithelial cells |
| CN104224842A (en) * | 2014-09-03 | 2014-12-24 | 苗九昌 | Preparation method of compound amniotic membrane powder and compound amniotic membrane powder prepared thereby |
Non-Patent Citations (1)
| Title |
|---|
| 廖琼等: "羊膜匀浆上清液对兔角膜上皮细胞增殖的影响", 《眼科新进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020157195A1 (en) | 2019-01-30 | 2020-08-06 | Univerza V Ljubljani | Procedure for the preparation of an amniotic membrane homogenate based antimicrobial agent |
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