CN1560235A - Non serum substratum for in vitro culture and amplification of cutaneous keratin cell - Google Patents
Non serum substratum for in vitro culture and amplification of cutaneous keratin cell Download PDFInfo
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- CN1560235A CN1560235A CNA2004100163679A CN200410016367A CN1560235A CN 1560235 A CN1560235 A CN 1560235A CN A2004100163679 A CNA2004100163679 A CN A2004100163679A CN 200410016367 A CN200410016367 A CN 200410016367A CN 1560235 A CN1560235 A CN 1560235A
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Abstract
The invention discloses a serum-free culture medium used for culturing and amplifying skin keratinocytes in vitro. It adds amino acid, vitamin, hormone, growth gene, trace elements, and antioxidant, thus able to accelerate cell breeding and delay consenescence of the keratinocytes and makes them amplified in vitro by more times. Using the culture medium to make primary culture and subculture needs no trophoblast cells and no extracellular substrates like collagen, etc prepaved at the bottom of the culture bottle to help the keratinocytes adhere to the wall and grow.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of substratum, relate in particular to a kind of serum free medium that is used for separating the specific chemical components of the epidermal keratinocyte vitro culture obtain and amplification from skin histology.
Background technology
Skin burn patient and other dermatosis patient all were by auto-skin grafting or corpse dermatoplasty in the past, and the skin source is very limited.Along with development of biology, can use method of tissue engineering external structure artificial skin at present, thereby solve the problem in skin source.
Skin histology makes up needs a large amount of seed cells, and wherein the vitro culture of epidermal keratinocyte is a difficult point.Keratinocyte is a kind of committed stem cell, is easy to old and feeble in the vitro culture process and differentiation, thereby influences cell proliferation; Secondly because the change of growing environment, separate the keratinocyte obtain from skin histology and lost intravital three-dimensional environment external, the support and the synergy of various kinds of cell have been lost, present in addition vitro culture environment is difficult to living environment in the analog cell body, therefore is difficult to obtain a large amount of seed cells.So how culture environment, the differentiation that delays keratinocyte and aging outside the control agent in the vitro culture process, making it at the more multiple of amplification in vitro is that the external extensive amplification of keratinocyte need solve " bottleneck " problem.
Since Rheinwald in 1975 and Green vitro culture keratinocyte are succeedd, a series of culture systems and substratum that are used for keratinocyte vitro culture and amplification have been researched and developed, but these culture systems mostly need be with the 3T3 cell as trophoderm [Rheinwald, J.G., et al., Nature, 265, p.421-424 (1977)], or overlay extracellular matrix [Gilchrest in culture dish bottom, J.CellPhysiol.112, p.197-206 (1982)], and often to add the indefinite albumen of serum and chemical ingredients (as: ox pituitary gland extract in the substratum, Toxins,exo-, cholera etc.) [Johnson, E.W, et al., Invitro Cell.Dev.Biol.28 (A), p.429-435 (1992); Auger, F.A., et al., In vitro Cell.Dev.Biol.31, p.432-439 (1995)], with support and stimulate keratinocyte adherent, grow and keep normal cellular form and protein excretion ability.But there is variety of problems in the keratinocyte with these culture systems and culture medium culturing in fundamental research and clinical application: add serum in the substratum, cause fibroblastic pollution on the one hand, influence Keratiocyte growth, the differentiation of accelerator angle cell plastid, on the other hand because the not clear proteic existence of serum and composition, fundamental researchs such as pair cell biology, toxicology, pathology (as: research cytokine, medicine to the influence of Keratiocyte growth and differentiation etc.) form and disturb; The artificial skin tissue that adopts above-mentioned culture systems and substratum to make up simultaneously carries out transplanting in the body, may cause immune response or introduce unsafe factor (as: virus or other pathogenic agent etc.), therefore needs the serum free medium of exploitation specific chemical components.
In order to develop a kind of fill-in that need not to add uncertain composition, and can support the keratinocyte clone to form, the substratum of growth, people have studied various basic mediums, trace element, multiple hormone and somatomedin etc. are to the influence of Keratiocyte growth, 199, on the basis of substratum such as F12, regulate the composition of substratum by optimizing clonal growth, constituted new substratum: MCDB151, energy backer's epidermal keratinocyte growth behind the additional 1.0mg/mL foetal calf serum albumen (high molecular weight protein) in this substratum, but feeder layer technique for former be commissioned to train support be still essential, and the lower [Peehl of cloning efficiency that goes down to posterity and cultivate, D.M., et al., In Vitro, Vol.16 (6), pp.526-538 (1980)].
After this, document [Tsao, M.C., et al., J.Cell.Physiol., 110, pp.219-229 (1982)] report, in MCDB151, add trace element, invented a kind of new substratum---MCDB152, in this new substratum, further added Urogastron, Transferrins,iron complexes, hydrocortisone, thanomin, phosphorylethanolamine, Progesterone, magnesium and strontium.The effect of thanomin and phosphorylethanolamine is to replace ox pituitary gland extract.Former being commissioned to train when supporting, keratinocyte is transferred among the MCDB152 that contains additive, though there is not uncertain composition in the substratum, keratinocyte still can be grown, but can not further go down to posterity, and cloning efficiency is very low, have only about 2%, the cell that obtains can't freezing and recovery.Still require to use the trophocyte when supporting former being commissioned to train in addition.Behind MCDB152, document [Boyce, S.C., Ham, R.G., J.Invest.Dermatol.81, pp.33S-40S (1983); J.Tiss.Cult.Meth.9, pp.83-93 (1985)] disclosed by Boyce and the further calcium ion concn that improves wherein of Ham, and definite designation is the MCDB153 substratum, but MCDB153 neither an outstanding keratinocyte serum free medium, its limitation shows the limited in one's ability of breeding and amplifying cells, converge if keratinocyte grows to fully in MCDB153, then will lose its ability that goes down to posterity fast.
Owing to have wretched insufficiency aspect the serum free medium composition, therefore when utilizing these culture medium culturing keratinocytes, have to add serum, pituitary gland extract, the Toxins,exo-, cholera of dialysing or use growth matrix etc., be difficult to really realize the external serum-free culture of keratinocyte, cell takes place aging in culturing process easily, thereby influences a large amount of amplifications of cell.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of serum free medium that the skin keratin cells in vitro is cultivated and increased that is used for, to overcome the above-mentioned defective that prior art exists.
Design of the present invention:
A kind of substratum is to be comprised that by various nutritive ingredients numerous one-tenth such as amino acid, carbohydrate, inorganic salt, VITAMIN, hormone, somatomedin, trace element are grouped into, the exhaustion of any nutritive substance all can be grown by restrictive cell in culturing process, can cause necrocytosis when situation is serious, if but nutritive substance surplus, in culture environment, also can accumulate a large amount of by products, the pair cell toxigenicity, and cause necrocytosis, therefore require that various nutritive ingredients must be balanced in the substratum, to satisfy cell growth, metabolism and to keep the demand of function.
Keratinocyte belongs to a kind of stem cell, in the vitro culture process, be easy to differentiation and old and feeble, influencing cell increases in a large number, various composition pair cells in the substratum are aging to be had a significant impact, therefore in the keratinocyte substratum, add the composition that some delay cell senescence, promote cell proliferation, can prolong cell at external incubation time and improve multiplication capacity.For example: oxidative damage is an important factor that causes cell aging, in existing keratinocyte substratum, all do not add antioxidant [US Pat.No.5712163, US Pat.No.4940666], the present invention is by adding antioxidant, delayed the aging of keratinocyte to a certain extent, made it at the more multiple of amplification in vitro.
The present invention is used for the serum free medium that the skin keratin cells in vitro is cultivated and increased, and it is characterized in that this serum free medium is a kind of aqueous solution, comprises basic medium and following component:
Basic medium 12-18g/L
Histidine 0.1-1.0mmol/L
Isoleucine 0.1-0.6mmol/L
Tryptophane 0.05-0.3mmol/L
Phenylalanine 0.3-0.6mmol/L
Methionin 0.7-1.2mmol/L
Methionine(Met) 0.05-0.3mmol/L
Tyrosine 0.3-0.6mmol/L
Keratin cell growth factor 2 0-200ng/L and/or Urogastron 5-20
ng/L
Pantothenate 0.02-0.04mmol/L
Choline chloride 60 0.1-0.5mmol/L
Folic acid 0.01-0.03mmol/L
Inose 0.1-0.4mmol/L
Niacinamide 0.05-0.5mmol/L
Vitamin B6 0.02-0.06mmol/L
Riboflavin 0.001-0.01mmol/L
Thiamines 0.01-0.03mmol/L
Regular Insulin 1-20 μ g/L
Transferrins,iron complexes 1-20 μ g/L
Thanomin 0.01-0.05mmol/L
Phosphorylethanolamine 0.01-0.05mmol/L
Strontium chloride 1-5mmol/L
Bovine serum albumin 5-20 μ g/L
Hydrocortisone 0.1-1 μ g/L
Triiodo Tiroidina amine 10-40pmol/L
Calcium chloride 0.03-0.5mmol/L
Magnesium chloride 5-10mmol/L
Antioxidant is an amount of
Penicillin 50-200U/L
Streptomycin sulphate 50-200U/L
Said basic medium comprises MCDB153, no calcium DMEM or does not have calcium DMEM and Ham ' s F12 is 1: 0.3～3 mixture with volume ratio.
Said antioxidant comprises mercaptoethanol 0.01-0.05mmol/L and/or catalase 50-150U/L and/or superoxide-dismutase 50-150U/L and/or Sodium Selenite 0.01-0.05mmol/L;
Said MCDB153 is at document [Boyce, S.C., Ham, R.G., J.Invest.Dermatol.81, pp.33S-40S (1983); J.Tiss.Cult.Meth.9, pp.83-93 (1985)] and the products catalogue of SIGMA company in existing open report;
Said no calcium DMEM is existing open report in the products catalogue of SIGMA company;
Said Ham ' s F12 is existing open report in the products catalogue of SIGMA company;
The preparation method of substratum of the present invention is very easy, and the component of being addressed is stirred at normal temperatures, and dissolving mixes, and after 0.2 μ m filtering membrane filtration sterilization, promptly can be used for keratinocyte and cultivates.
Utilizing substratum of the present invention that keratinocyte is carried out former being commissioned to train supports and the cultivation of going down to posterity, owing to add amino acid, VITAMIN, hormone, somatomedin, trace element and antioxidant, do not need the trophocyte, also need not to overlay extracellular matrix such as collagen protein and help the adherent and growth of keratinocyte in culturing bottle bottom, promote simultaneously keratinocyte propagation to a certain extent, delaying cell aging makes it at the more multiple of amplification in vitro.
Description of drawings
Fig. 1 is cultured to converge keratinocyte in former generation.
Fig. 2 has compared the growth curve of mouse keratinocyte in serum free medium of the present invention and traditional substratum.
Embodiment
Embodiment 1
As basic medium, its component is as follows with MCDB153: (in 1L)
MCDB153 17.61g
Histidine 0.3mmol
Isoleucine 0.6mmol
Tryptophane 0.2mmol
Phenylalanine 0.3mmol
Methionin 0.7mmol
Methionine(Met) 0.2mmol
Tyrosine 0.3mmol
Calcium chloride 0.1mmol
Magnesium chloride 5mmol
Keratinocyte growth factor 100ng
Pantothenate 0.021mmol
Choline chloride 60 0.171mmol
Folic acid 0.024mmol
Inose 0.211mmol
Niacinamide 0.082mmol
Vitamin B6 0.049mmol
Riboflavin 0.003mmol
Thiamines 0.03mmol
Sigma I8405 5 μ g
Change iron egg 10 μ g
Thanomin 0.05mmol
Strontium chloride 1mmol
Bovine serum albumin 10 μ g
Hydrocortisone 0.1 μ g
Triiodo Tiroidina amine 40pmol
Mercaptoethanol 0.05mmol
Penicillin: 100U
Streptomycin sulphate: 100U
Said components is dissolved in the 1L water, mixes, be the said substratum of the present invention.After 0.2 μ m filtering membrane filtration sterilization, can be used for keratinocyte and cultivate.
Embodiment 2
Get newborn 1 day SD rat skin or people's foreskin, be cut into 1cm
2About skin chunk, clean 3 times with the PBS that contains the 200U/mL gentamicin, 75% alcohol wash 2 times is cleaned 2 times with PBS again.Skin histology is joined in the neutral protease of 200U/mL, 4 ℃ of digestion 18hr, corium is separated with epidermis, then epidermis is shredded, pancreatin/0.02%EDTA with 0.025% is 6mL effect 5min altogether, the centrifugal 5min of 2000rpm, and add among the 10mg/mL soybean pancreatin inhibitor 2-3mL and the pancreatin activity.150 eye mesh screens filter, and remove the skin fragment.The centrifugal 5min of 2000rpm removes supernatant, and PBS cleans 2 times, adds the keratinocyte serum free medium of embodiment 1.The cell inoculation amount is 2 * 10
4Cells/cm
2, each 25cm
2Culturing bottle in add substratum 5mL, at 37 ℃, 5%CO
2Incubator in cultivate, changed liquid once in per three days.Keratinocyte after covering with culturing bottle bottom 70%-80% in 7-10 days, the cultivation of can going down to posterity.Accompanying drawing 1 is to utilize serum free medium of the present invention to cultivate former generation keratinocyte to the photo that converges.
Embodiment 3
Former keratinocyte of being commissioned to train after supporting adds 3mL 0.025% pancreatin/0.02%EDTA to be digested, and when cell rounding, the back centrifugal 5min of 2000rpm that comes off, abandons Digestive system, adds in the 2-3mL 10mg/mL soybean pancreatin inhibitor and the pancreatin activity.Remove supernatant behind the centrifugal 5min of 2000rpm, PBS cleans twice, with 2 * 10
4The inoculation of cells/mL inoculum size, each culturing bottle adds serum free medium 5mL involved in the present invention, the cultivation of going down to posterity.Accompanying drawing 2 has compared the growth curve of mouse keratinocyte in serum free medium of the present invention and traditional substratum.Serum free medium of the present invention adopts the substratum of embodiment 1.
Embodiment 4
Keratinocyte is the more intense cell of a kind of differentiation capability, and its atomization is divided into a plurality of stages, and generally (Cross-linked Envelope, the terminal stage of its differentiation is thought in formation CE) the crosslinked capsid protein of keratinocyte.With cultured cells in 0.25% (w/v) pancreatin solution digestion square vase, PBS cleans back meter cell count, the centrifugal then supernatant liquor of abandoning, adding 5mL contains the PBS solution of 1% sodium lauryl sulphate (SDS), 20mmol/L dithiothreitol (DTT) (DTT), 90 ℃ of water-baths 10 minutes, centrifugal, meter capsid protein number.Keratinocyte capsid protein ratio=(capsid protein number/total cell count) * 100%.Old and feeble keratinocyte can be differentiated by the beta-galactosidase enzymes stained positive.In the cultivation latter stage in per generation, cell washes twice with the PBS damping fluid, at room temperature uses 3% formalin fixed then
Minute.Clean with PBS damping fluid and distilled water again, add freshly prepared
The tilactase staining fluid: 1mg/mL X-Gal, the 40mM citric acid, the 12mM sodium phosphate, the 5mM yellow prussiate of potash, the 5mM Tripotassium iron hexacyanide, 150mM sodium-chlor, the 2mM magnesium chloride spends the night under 37 ℃.The ratio of counting cells Smalt cell, 700 cells of minimum counting in each sample are be exactly total the ratio blue cell of senile cell accounts for the ratio of cell concentration.Subordinate list 1 has compared the human skin keratinocyte in the serum free medium of embodiment 1 and differentiation and the aging conditions in traditional substratum.
Differentiation and the aging conditions of table 1. human skin keratinocyte in serum free medium of the present invention and traditional substratum
Substratum senile cell ratio (%) noble cells ratio (%)
Traditional serum free medium 48 ± 5 39 ± 1
Substratum 33 ± 3 28 ± 2 of embodiment 1
Traditional serum free medium adopts [Yang, E.K., et al., Artificial Organs, 24 (1), pp7-17 (2000); Hansbrough, J.F., Epidermal Cells.In:Ricordi, C., eds.Methods in cell transplantation.Austin:R.G.Landes Company Press, pp111-121 (1995)] the disclosed keratinocyte serum free medium of document.
Claims (4)
1. be used for the serum free medium that the skin keratin cells in vitro is cultivated and increased, it is characterized in that being a kind of aqueous solution, comprise basic medium and following component:
Basic medium 12-18g/L
Histidine 0.1-1.0mmol/L
Isoleucine 0.1-0.6mmol/L
Tryptophane 0.05-0.3mmol/L
Phenylalanine 0.3-0.6mmol/L
Methionin 0.7-1.2mmol/L
Methionine(Met) 0.05-0.3mmol/L
Tyrosine 0.3-0.6mmol/L
Keratin cell growth factor 2 0-200ng/L and/or Urogastron 5-20ng/L
Pantothenate 0.02-0.04mmol/L
Choline chloride 60 0.1-0.5mmol/L
Folic acid 0.01-0.03mmol/L
Inose 0.1-0.4mmol/L
Niacinamide 0.05-0.5mmol/L
Vitamin B6 0.02-0.06mmol/L
Riboflavin 0.001-0.01mmol/L
Thiamines 0.01-0.03mmol/L
Regular Insulin 1-20 μ g/L
Transferrins,iron complexes 1-20 μ g/L
Thanomin 0.01-0.05mmol/L
Phosphorylethanolamine 0.01-0.05mmol/L
Strontium chloride 1-5mmol/L
Noon serum albumin 5-20 μ g/L
Hydrocortisone 0.1-1 μ g/L
Triiodo Tiroidina amine 10-40pmol/L
Calcium chloride 0.03-0.5mmol/L
Magnesium chloride 5-10mmol/L
Antioxidant is an amount of
Penicillin 50-200U/L
Streptomycin sulphate 50-200U/L
2. the serum free medium that is used for cultivation of skin keratin cells in vitro and amplification according to claim 1, it is characterized in that said basic medium comprises MCDB153, no calcium DMEM or do not have calcium DMEM and Ham ' s F12 volume ratio is 1: 0.3～3 mixture.
3. the serum free medium that is used for cultivation of skin keratin cells in vitro and amplification according to claim 1, it is characterized in that said antioxidant comprises mercaptoethanol 0.01-0.05mmol/L and/or catalase 50-150U/L and/or superoxide-dismutase 50-150U/L and/or Sodium Selenite 0.01-0.05mmol/L.
4. the serum free medium that is used for cultivation of skin keratin cells in vitro and amplification according to claim 2, it is characterized in that said antioxidant comprises mercaptoethanol 0.01-0.05mmol/L and/or catalase 50-150U/L and/or superoxide-dismutase 50-150U/L and/or Sodium Selenite 0.01-0.05mmol/L.
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| CN105219697A (en) * | 2014-07-04 | 2016-01-06 | 赫柏慧康生物科技无锡有限公司 | A kind of people's primary keratinocyte is separated the method for preparation |
| CN105462916A (en) * | 2015-12-22 | 2016-04-06 | 肇庆大华农生物药品有限公司 | Serum-free medium for culturing Marc-145 cell and preparation method thereof |
| CN106047793A (en) * | 2016-06-16 | 2016-10-26 | 广东科玮生物技术股份有限公司 | Culture medium for skin keratinocyte and culture method thereof |
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| CN111876373A (en) * | 2020-08-04 | 2020-11-03 | 任涛 | Serum-free culture medium with definite chemical components for human airway epithelial cells and application thereof |
| CN112980775A (en) * | 2021-03-19 | 2021-06-18 | 上海爱萨尔生物科技有限公司 | Culture solution for preparing keratinocytes based on differentiation of pluripotent stem cells |
| CN113046300A (en) * | 2021-03-19 | 2021-06-29 | 上海爱萨尔生物科技有限公司 | Culture method for preparing keratinocytes based on differentiation of pluripotent stem cells |
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| CN105219697A (en) * | 2014-07-04 | 2016-01-06 | 赫柏慧康生物科技无锡有限公司 | A kind of people's primary keratinocyte is separated the method for preparation |
| CN105462916A (en) * | 2015-12-22 | 2016-04-06 | 肇庆大华农生物药品有限公司 | Serum-free medium for culturing Marc-145 cell and preparation method thereof |
| CN105462916B (en) * | 2015-12-22 | 2018-04-13 | 肇庆大华农生物药品有限公司 | A kind of serum free medium of culture 145 cells of Marc and preparation method thereof |
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| CN106047793B (en) * | 2016-06-16 | 2019-08-13 | 广东科玮生物技术股份有限公司 | A kind of skin keratin forms the culture medium and its cultural method of cell |
| CN106520674A (en) * | 2016-12-24 | 2017-03-22 | 严志海 | Serum-free medium for in-vitro culture of epidermal melanocytes |
| CN107312745B (en) * | 2017-06-18 | 2023-03-03 | 广东博溪生物科技有限公司 | Serum-free epithelial cell culture solution |
| CN107287150A (en) * | 2017-06-18 | 2017-10-24 | 广东博溪生物科技有限公司 | A kind of construction method of 3D epidermises model |
| CN107312745A (en) * | 2017-06-18 | 2017-11-03 | 广东博溪生物科技有限公司 | A kind of epithelial cell nutrient solution of serum-free |
| CN107287150B (en) * | 2017-06-18 | 2020-08-04 | 广东博溪生物科技有限公司 | Construction method of 3D (three-dimensional) epidermis model |
| CN108410800A (en) * | 2018-03-26 | 2018-08-17 | 郭子宽 | It is a kind of culture human amnion membrane culture medium and application |
| CN109234223A (en) * | 2018-11-21 | 2019-01-18 | 南京基蛋生物医药有限公司 | Low albumen serum-free cell culture medium |
| CN109234223B (en) * | 2018-11-21 | 2021-01-19 | 南京基蛋生物医药有限公司 | Low-protein serum-free cell culture medium |
| CN111876373A (en) * | 2020-08-04 | 2020-11-03 | 任涛 | Serum-free culture medium with definite chemical components for human airway epithelial cells and application thereof |
| CN112980775A (en) * | 2021-03-19 | 2021-06-18 | 上海爱萨尔生物科技有限公司 | Culture solution for preparing keratinocytes based on differentiation of pluripotent stem cells |
| CN113046300A (en) * | 2021-03-19 | 2021-06-29 | 上海爱萨尔生物科技有限公司 | Culture method for preparing keratinocytes based on differentiation of pluripotent stem cells |
| CN112980775B (en) * | 2021-03-19 | 2024-01-23 | 上海爱萨尔生物科技有限公司 | Culture solution for preparing keratinocyte based on differentiation of pluripotent stem cells |
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