CN106367388A - Method for protecting umbilical cord mesenchymal stem cell culture supernatant at 4 DEG C - Google Patents

Method for protecting umbilical cord mesenchymal stem cell culture supernatant at 4 DEG C Download PDF

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CN106367388A
CN106367388A CN201611023963.9A CN201611023963A CN106367388A CN 106367388 A CN106367388 A CN 106367388A CN 201611023963 A CN201611023963 A CN 201611023963A CN 106367388 A CN106367388 A CN 106367388A
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umbilical cord
stem cell
culture supernatant
supernatant
culture
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CN106367388B (en
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楼敏铭
马洁
程腾
孟欢
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TIANJIN KANGTING BIOTECHNOLOGY Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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Abstract

The invention relates to a method for protecting umbilical cord mesenchymal stem cell culture supernatant at 4 DEG C. The method includes the steps that firstly, a full-term umbilical cord is taken, wherein the full-term umbilical cord needs to be authenticated before being used; then, culture and starvation culture are carried out, supernatant is prepared, and a protective agent is added; finally, cold storage is carried out. By means of the method for protecting the umbilical cord mesenchymal stem cell culture supernatant at 4 DEG C, it can be ensured that the protein content of the stem cell supernatant does not change after the umbilical cord mesenchymal stem cell culture supernatant is stored for 15 days at 4 DEG C, the storage time of the stem cell culture supernatant at 4 DEG C is prolonged, and the transport cost of the stem cell culture supernatant can be greatly reduced.

Description

The 4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant
Technical field
The invention belongs to biological technical field, it is related to a kind of 4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant.
Background technology
Mescenchymal stem cell (mscs) is found in nineteen sixty-eight, the first passage in bone marrow such as friedenstein aj earliest Adherent method obtains a kind of fibroblast, further study show that, mscs be initiated by mesoderm have Multidirectional Differentiation and The adult stem cell of self-renewal capacity, current mscs's is tissue-derived a lot, including bone marrow, fatty tissue, skin histology, umbilicuss Band blood and umbilical cord tissue etc..
Umbilical cord mesenchymal stem cells derive from neonate and discard umbilical cord tissue, have powerful self-renewal capacity and to fat Fat cell, neurocyte, hepatocyte and myocyte's differentiation potential, because of its abundance it is easy to obtain, and obtain hurtless measure in addition Immunogenicity is low, and multiplication capacity is strong, and no matter the advantage such as rationality dispute, is widely used in various clinical researches.
The umbilical cord mesenchymal stem cells secretory component of report is broadly divided into 3 classes: 1, extracellular matrix class at present, such as various Collagen protein and elastin laminin etc., can promote reinventing of extracellular matrix；2nd, somatomedin class, such as tgf, hgf, vegf and bfgf Deng；3rd, inflammatory factor and chemotactic factor class, such as pge2, il-6, il-8 etc..Due to umbilical cord mesenchymal stem cells can secrete multiple The factor, therefore, increasing research concentrates on the paracrine action of stem cell, and research shows, stem cell paracrine action pair Skin injury is repaired, and participates in the aspects such as the immune response of body and has important effect.
Multiple factors of umbilical cord mesenchymal stem cells secretion mostly are protein ingredient, how to guarantee in stem cell supernatant effectively because The content of son, is current urgent problem, is also to directly affect one of bottleneck of stem cell clinical practice, extends egg at present The only effective method of white holding time is to reduce to preserve temperature DEG C, and conventional is that less than -20 DEG C temperature DEG C preserve, and this also gives albumen The transport of class material increased cost, the present invention based on extend umbilical cord mesenchymal stem cells culture supernatant resting period, really Protect the content of stem cell secretion factor, providing for Clinical practice stem cell secretion factor may.
Content of the invention
It is an object of the invention in place of overcoming the deficiencies in the prior art, providing in a kind of umbilical cord mesenchymal stem cells culture Clear 4 DEG C of guard methods, this method extends 4 DEG C of resting periods of umbilical cord mesenchymal stem cells culture supernatant.
The technical solution used in the present invention is:
A kind of 4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant, step is as follows:
(1) take fully a moon healthy fetus umbilical cord, detect cord serum with the method that gold colloidal, fluorescent quantitation pcr combine Hbsag, anti-hcv, hcv-rna, anti-hdv, anti-hev, anti-hiv-1/2, hiv-1-rna, cmv-dna, testing result is the moon The umbilical cord of property can use；
(2) umbilical cord is placed in sterilized petri dishes, is cut into the segment of 5cm with aseptic operation, is repeatedly cleaned with pbs and remain in Endovascular blood, till the pbs after cleaning is substantially colorless；
(3) the umbilical cord after cleaning is transferred in kidney basin, after peeling off umbilical vein and umbilical artery, with operating scissorss, umbilical cord is irrigated and time Glue cuts into the fritter of 1-5mm, evenly laid out to t75 culture bottle bottom；
(4) the culture bottle of the umbilical cord tissue block that tiled is put in CO2 gas incubator, be dried 30 minutes；
(5) it is slowly added to the culture medium containing 10% hyclone and serum albumin additive toward in culture bottle, culture medium Consumption is advisable with just submergence piece of tissue；
(6) change within every 6 days liquid 1 time, tissues observed paste block border cell's growing state, treat most of piece of tissue cells climb out of and Cell growth around each block organization's block, to during 80% about fusion, carries out the primary of umbilical cord mesenchymal stem cells and passes on；
(7) press 8000cells/cm2Density inoculation p4 for umbilical cord mesenchymal stem cells cell in t75 culture bottle, add 10ml df12 complete medium；
(8) when cell length to 80% merges, with 5mlpbs wash buffer cell surface 3 times；
(9) exhaust pbs buffer, add the hungry culture of 10mldf12 culture medium 72 hours；
(10) collect the umbilical cord mesenchymal stem cells culture supernatant after hungry culture 72 hours, supernatant is collected 50ml centrifugation 300g in pipe, room temperature is centrifuged 5 minutes；
(11) abandon precipitation, collect stem cell culture supernatant, with the bag filter of 5kd, stem cell supernatant is concentrated, concentrate 10 times, obtain the effective stem cell secretion hybrid cytokine containing more than 5kd molecular weight；
(12) in stem cell culture supernatant after concentration, add the Mannitol of 10 volume ratio % glycerol or 10%g/ml, as Stem cell culture supernatant protective agent, puts into 4 DEG C of Refrigerator stores using without protectant stem cell culture supernatant as comparison.
And, described step (12) in stem cell culture supernatant in add 5% volume ratio glycerol, after incubation 30 minutes, then Add the Mannitol of 5%g/ml.
The invention has the benefit that
4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant that the present invention provides add interpolation protection in cold-storage 4 DEG C of times deposited of stem cell supernatant can be extended, providing for clinical practice stem cell secretion factor may after agent.
4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant that the present invention provides guarantee 4 DEG C deposit 15 days after, do thin Protein content in born of the same parents' supernatant does not change, and extends the resting period of 4 DEG C of stem cell culture supernatant, can substantially reduce stem cell training The cost of transportation of foster supernatant.
The protective agent adding, glycerol or Mannitol do not affect the molecular structure of supernatant composition and composition, and as skin During beauty treatment, certain effect can also be strengthened to a certain extent.
Specific embodiment
With reference to specific embodiment, the invention will be further described, and following examples are descriptive, is not limit Qualitatively it is impossible to protection scope of the present invention is limited with this.
A kind of 4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant, step is as follows:
(1) take fully a moon healthy fetus umbilical cord, detect cord serum with the method that gold colloidal, fluorescent quantitation pcr combine Hbsag, anti-hcv, hcv-rna, anti-hdv, anti-hev, anti-hiv-1/2, hiv-1-rna, cmv-dna, testing result is the moon The umbilical cord of property can use；
(2) umbilical cord is placed in sterilized petri dishes, with aseptic operation be cut into 5cm about segment, repeatedly cleaned residual with pbs Stay endovascular blood, till the pbs after cleaning is substantially colorless；
(3) the umbilical cord after cleaning is transferred in kidney basin, after peeling off umbilical vein and umbilical artery, with operating scissorss, umbilical cord is irrigated and time Glue cuts into the fritter of 1-5mm, evenly laid out to t75 culture bottle bottom；
(4) the culture bottle of the umbilical cord tissue block that tiled is put in CO2 gas incubator, be dried 30 minutes；
(5) it is slowly added to the culture medium containing 10% hyclone and serum albumin additive toward in culture bottle, culture medium Consumption is advisable with just submergence piece of tissue；
(6) change within every 6 days liquid 1 time, tissues observed paste block border cell's growing state, treat most of piece of tissue cells climb out of and Cell growth around each block organization's block, to during 80% about fusion, carries out the primary of umbilical cord mesenchymal stem cells and passes on；
(7) press 8000cells/cm2Density inoculation p4 for umbilical cord mesenchymal stem cells cell in t75 culture bottle, add 10ml df12 complete medium；
(8) when cell length to 80% about merges, with 5mlpbs wash buffer cell surface 3 times；
(9) exhaust pbs buffer, add the hungry culture of 10mldf12 culture medium 72 hours；
(10) collect the umbilical cord mesenchymal stem cells culture supernatant after hungry culture 72 hours, supernatant is collected 50ml centrifugation 300g in pipe, room temperature is centrifuged 5 minutes；
(11) abandon precipitation, collect stem cell culture supernatant, with the bag filter of 5kd, stem cell supernatant is concentrated, concentrate 10 times, obtain the effective stem cell secretion hybrid cytokine containing more than 5kd molecular weight；
(12) in stem cell culture supernatant after concentration, add the Mannitol of 10 volume ratio % glycerol or 10%g/ml, as Stem cell culture supernatant protective agent, puts into 4 DEG C of Refrigerator stores using without protectant stem cell culture supernatant as comparison；
(14) the concentration of albumen in each group stem cell supernatant after depositing 15 days, is measured with cck-8.
The present invention step (12) in stem cell culture supernatant in add 5% volume ratio glycerol, after incubation 30 minutes, then Add the Mannitol of 5%g/ml, as stem cell culture supernatant protective agent, to make without protectant stem cell culture supernatant For comparison, place into 4 DEG C of Refrigerator stores.
Table 1: the protein content in stem cell culture supernatant

Claims (2)

1. a kind of 4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant it is characterised in that: step is as follows:
(1) take fully a moon healthy fetus umbilical cord, detect cord serum hbsag with the method that gold colloidal, fluorescent quantitation pcr combine, Anti- hcv, hcv-rna, anti-hdv, anti-hev, anti-hiv-1/2, hiv-1-rna, cmv-dna, testing result is the umbilicuss of feminine gender Band can use；
(2) umbilical cord is placed in sterilized petri dishes, is cut into the segment of 5cm with aseptic operation, is repeatedly cleaned with pbs and remain in blood vessel Interior blood, till the pbs after cleaning is substantially colorless；
(3) the umbilical cord after cleaning is transferred in kidney basin, after peeling off umbilical vein and umbilical artery, with operating scissorss, umbilical cord jelly of Wharton is cut Be cut into the fritter of 1-5mm, evenly laid out to t75 culture bottle bottom；
(4) the culture bottle of the umbilical cord tissue block that tiled is put in CO2 gas incubator, be dried 30 minutes；
(5) it is slowly added to the culture medium containing 10% hyclone and serum albumin additive, the consumption of culture medium toward in culture bottle It is advisable with just submergence piece of tissue；
(6) change within every 6 days liquid 1 time, tissues observed paste block border cell's growing state, treat that most of piece of tissue cells climb out of and each Cell growth around block organization's block, to during 80% about fusion, carries out the primary of umbilical cord mesenchymal stem cells and passes on；
(7) press 8000cells/cm2Density inoculation p4 for umbilical cord mesenchymal stem cells cell in t75 culture bottle, add 10ml Df12 complete medium；
(8) when cell length to 80% merges, with 5ml pbs wash buffer cell surface 3 times；
(9) exhaust pbs buffer, add the hungry culture of 10ml df12 culture medium 72 hours；
(10) collect the umbilical cord mesenchymal stem cells culture supernatant after hungry culture 72 hours, supernatant is collected in 50ml centrifuge tube 300g, room temperature is centrifuged 5 minutes；
(11) abandon precipitation, collect stem cell culture supernatant, with the bag filter of 5kd, stem cell supernatant is concentrated, concentrate 10 times, Obtain the effective stem cell secretion hybrid cytokine containing more than 5kd molecular weight；
(12) add the Mannitol of 10 volume ratio % glycerol or 10%g/ml in stem cell culture supernatant after concentration, as dry thin Born of the same parents' culture supernatant protective agent, puts into 4 DEG C of Refrigerator stores using without protectant stem cell culture supernatant as comparison.
2. 4 DEG C of guard methods of umbilical cord mesenchymal stem cells culture supernatant according to claim 1 it is characterised in that: described Step (12) in stem cell culture supernatant in add 5% volume ratio glycerol, after incubation 30 minutes, add the manna of 5%g/ml Alcohol.
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CN106880645A (en) * 2017-02-28 2017-06-23 天津普瑞赛尔生物科技有限公司 Enema liquid containing mescenchymal stem cell secretion for small household pet and preparation method thereof
CN108103014A (en) * 2017-12-28 2018-06-01 重庆斯德姆生物技术有限公司 A kind of method for preserving of umbilical cord mesenchymal stem cells culture supernatant
CN108496957A (en) * 2018-06-04 2018-09-07 北京中广天生物科技有限公司 The deepfreeze store method of umbilical cord mesenchymal stem cells culture supernatant
CN108517002A (en) * 2018-04-20 2018-09-11 命之本源医疗科技（北京）有限公司 A kind of stabilizer of stem cell culture supernatant secretory protein
CN108753706A (en) * 2018-06-04 2018-11-06 北京中广天生物科技有限公司 Application of the white flower loropetalum chinense in preparing umbilical cord mesenchymal stem cells culture supernatant
CN112640890A (en) * 2020-12-30 2021-04-13 重庆市铂而斐细胞生物技术有限公司 Cell preserving fluid for delaying cell growth in cell culture and preparation method thereof

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106880645A (en) * 2017-02-28 2017-06-23 天津普瑞赛尔生物科技有限公司 Enema liquid containing mescenchymal stem cell secretion for small household pet and preparation method thereof
CN108103014A (en) * 2017-12-28 2018-06-01 重庆斯德姆生物技术有限公司 A kind of method for preserving of umbilical cord mesenchymal stem cells culture supernatant
CN108517002A (en) * 2018-04-20 2018-09-11 命之本源医疗科技（北京）有限公司 A kind of stabilizer of stem cell culture supernatant secretory protein
CN108496957A (en) * 2018-06-04 2018-09-07 北京中广天生物科技有限公司 The deepfreeze store method of umbilical cord mesenchymal stem cells culture supernatant
CN108753706A (en) * 2018-06-04 2018-11-06 北京中广天生物科技有限公司 Application of the white flower loropetalum chinense in preparing umbilical cord mesenchymal stem cells culture supernatant
CN112640890A (en) * 2020-12-30 2021-04-13 重庆市铂而斐细胞生物技术有限公司 Cell preserving fluid for delaying cell growth in cell culture and preparation method thereof

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