CN109055303A - A kind of construction method of skin histology - Google Patents
A kind of construction method of skin histology Download PDFInfo
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- CN109055303A CN109055303A CN201811060758.9A CN201811060758A CN109055303A CN 109055303 A CN109055303 A CN 109055303A CN 201811060758 A CN201811060758 A CN 201811060758A CN 109055303 A CN109055303 A CN 109055303A
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The present invention relates to field of cell culture, in particular to a kind of construction method of skin histology, comprising the following steps: the Green culture solution of above-mentioned optimization is added in the bracket of three-dimensional streaming bioreactor in fibroblast kind in bracket；RPMI1640 culture solution is added in branch frame peripheral, cultivates 90-110h, keratinocyte, culture is added in the rack surface；Gas-liquid interface is formed, culture solution is regularly replaced, continues culture 21 ± 1 days.The present invention passes through three-dimensional streaming bioreactor for fibroblast and the common stereoscopic culture of keratinocyte, obtains the skin histology for meeting demand, which can be used for the transplanting of skin.
Description
Technical field
The present invention relates to field of cell culture, in particular to a kind of construction method of skin histology.
Background technique
Skin is the maximum organ of human body.The main reason for large area skin lacks is burn and scald.It unites according to nineteen fifty-two
Meter investigation, in 15-44 years old crowd, when 60% or more skin of the body surface gross area loses, mortality is 100% (Bull
And Fischer, 1954)；But the death rate dropped to 41.4% (Chua et al 2003) in 2003.What the death rate reduced
Main cause is the raising for the treatment of technology.At present treatment widespread skin missing treatment be mainly self transplantation of free skin graft and
Artificial synthesized dermatoplasty.
Artificial synthesized skin can timely succour patient, save life.And organization engineering skin cell compatibility is high, tool
There is more obvious advantage, is also more suitable for patient.But expensive is that patient is unacceptable, particularly in China.Each big face
Product dermatoplastic (required cost is at 500,000 dollars or more), limits the development and application of organization engineering skin at home.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of construction methods of skin histology, can turn out on the basis of lower price
The biggish full thickness skin of area, the skin histology have preferable biocompatibility simultaneously, meet the needs of patient.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of Green culture solution of optimization, DMEM culture medium and Ham ' s F12 nutrient solution are 3 ± 0.2:1 group by volume
At mixed liquor in following components is added: in terms of the volume of the mixed liquor, 10 ± 1ng/ml of EGF, hydrocortisone 0.4 ±
0.05 μ g/ml, 0.18 ± 0.02mM of adenine, 5 ± 0.5 μ g/ml of insulin, 5 ± 0.5 μ g/ml of transferrins, glutamine 2
± 0.2mM, 0.2 ± 0.05 μM of iodine thyronine, the percentage by volume of FCS and antibiotic, the FCS addition is described
The 10% ± 1% of mixeding liquid volume.
The Green culture solution of optimization provided by the invention, is related to the cultivating process of keratinocyte and skin histology, is
The cultivation of keratinocyte and skin histology provides good basis.
The range of each component is equally possible the error range being related to during the experiment, and therefore, each component is in the range
It is within the scope of the invention.
Antibiotic is added in Green culture solution mainly prevents pollution of the bacterium to culture medium, according to related bacterium
The different antibiotic of addition can be selected.
Further, the antibiotic includes that amphotericin B, penicillin, streptomysin are any one or more of.
Further, in terms of the volume of the mixed liquor, the additive amount of each antibiotic are as follows: amphotericin B 0.625 ± 0.05
μ g/ml, 100 ± 5U/ml of penicillin, 100 ± 5 μ g/ml of streptomysin.
The present invention also provides a kind of keratinocyte cultural methods, comprising the following steps:
Keratinocyte is resuspended in the Green culture solution of above-mentioned optimization, and auxiliary 3T3 cell, culture is added；
KSFM culture medium is replaced after 7-8 hours, and 10 ± 1ng/ml of EGF is added；
Every 3 days replacement culture solutions, PBS cleaning, culture obtain the keratinocyte.
KSFM culture medium is that (abbreviation of Keratinocyte Serum-free Growth medium is a kind of commercially available training
Support base.
Further, the number ratio of the keratinocyte and the 3T3 cell is 1:0.8-1.2.
Further, the keratinocyte is digested isolated by epidermal tissue.
Further, the epidermal tissue is isolated by thigh skin, preferably medial thigh skin.
Further, the time of the culture is 10-15 days, and cell converges up to 70%-90%, obtains the cutin and is formed
Cell.
Further, after the cell converges up to 70%-90%, further includes: diluting cells continue secondary culture, obtain
The keratinocyte.
The keratinocyte that i.e. present invention culture obtains can obtain more keratinocytes by secondary culture,
To meet actual quantity demand.
Material used in the present invention may be derived from autologous tissue, such as thigh skin, especially medial thigh skin, the skin taken
Skin can such as operate according to the following steps through enzymic digestion:
Under aseptic condition, medial thigh skin tissue is taken, is placed in and contains 10% newborn calf serum (FBS) and dual anti-
In the DMEM of (100 μ g/ml streptomysin of 100IU/ml penicillin), 4 DEG C of preservations.
Skin histology shearing, with the phosphate containing 50U/ml penicillin, 50g/ml streptomysin and 50g/ml gentamicin
It is cleaned for several times in buffer saline (PBS).
Tissue after cleaning is immersed in containing dual anti-dose (100U/ml penicillin and 100 μ g/ml streptomysins) and tryptose
The PBS mixed liquor of enzymic digestion liquid is kept for 4 DEG C of temperature, stood overnight.
Postdigestive skin is poured into culture dish, is separated epidermis and corium with tweezers, respectively obtain epidermal tissue and
Dermal tissue.
Isolated epidermal tissue obtains single cell suspension after enzymic digestion, and then isolated keratinocyte, obtains
Keratinocyte obtain keratinocyte through above-mentioned method culture.
The present invention also provides a kind of fibroblastic cultural methods, comprising the following steps:
Dermal tissue uses collagenase digesting, is isolated to fibroblast single cell suspension；
The fibroblast single cell suspension is added in the DMEM culture solution containing fetal calf serum and hydrocortisone,
In terms of the volume of the DMEM culture solution, the addition volume of the fetal calf serum is 10% ± 1%, the hydrocortisone
0.4μg/ml；
Culture, cultivates fluid exchange after 24-48 hours, then the culture solution of replacement in every 2-3 days, until 85%- for the first time
After 90% converges, the fibroblast is obtained.
Fibroblastic cultural method provided by the invention obtains more fibroblast by culture, should
Culture be originally culture, in order to obtain more fibroblasts can the fibroblast to originally culture carry out passage training
It supports.
Wherein, fibroblast can be used following steps and be made:
Dermal tissue obtained above is cleaned 3 times with PBS, is cut into the fragment of very little, fragment is transferred to containing dual anti-dose
It is digested in the centrifuge tube of clostridiopetidase A II type (200IU/ml), fibroblast single cell suspension is obtained by filtration.
Further, the culture are as follows: in 37 ± 2 DEG C and 5%CO2Lower culture cell.
Further, the dermal tissue is isolated by thigh skin, preferably medial thigh skin.
Further, the dermal tissue and above-mentioned epidermal tissue derive from same skin.
Further, the fibroblast obtained further includes the steps that secondary culture, obtains more described at fiber
Cell.The same originally culture of culture medium and condition of culture of secondary culture.
As operated according to the following steps:
Converging when fibroblast reaches 85%-90%, removes culture solution, tryptic digestive juice is added in PBS cleaning,
Digestion；
It is blown and beaten repeatedly with suction pipe after terminating digestion to obtain single cell suspension, supernatant, secondary culture, 37 are abandoned in centrifugation
DEG C and 5%CO2Lower culture cell, cultivates fluid exchange for the first time after 24-48 hours, then replacement in every 2-3 days is primary.
After 85%-90% converges, cell is spare, or is frozen in -150 DEG C of liquid nitrogen storage tanks at once.
By above-mentioned culture, more fibroblasts are obtained.
The present invention also provides a kind of construction methods of skin histology, comprising the following steps:
The Green of above-mentioned optimization is added in the bracket of three-dimensional streaming bioreactor in fibroblast kind in bracket
Culture solution；
RPMI1640 culture solution is added in branch frame peripheral, cultivates 90-110h, cutin is added in the rack surface and is formed carefully
Born of the same parents, culture；
Gas-liquid interface is formed, culture solution is regularly replaced, continues culture 21 ± 1 days.
The present invention passes through three-dimensional streaming bioreactor for fibroblast and the common stereoscopic culture of keratinocyte, obtains
To the skin histology for meeting demand, which can be used for the transplanting of skin.
Three-dimensional streaming bioreactor is researched and developed from German Mai Dekesi Bioisystech Co., Ltd used in the present invention 3
Streaming bioreactor is tieed up, the living environment of better simulated skin cell more easily controls the life condition of Skin Cell,
It is simultaneously that biological support is eliminated the hidden danger of rejection, cultivated using autogenous cell using biological carrier materials, that is, bracket
The full thickness skin equivalent equivalent in biological nature, physical mechanical characteristic with application on human skin.
Further, density of the fibroblast in the bracket is 5 × 105/cm2。
Further, the fibroblast is using fibroblast made from the above method.
Further, the additional amount of the keratinocyte is 1 × 106cells/ml。
Further, the keratinocyte is using keratinocyte made from the above method.
Further, the fibroblast and the keratinocyte derive from same skin histology, through separating
It obtains.It can be selected from autologous skin tissue, preferably leg inside skin.
Further, the condition cultivated in the construction method of skin histology is 37 ± 1 DEG C, 5%CO2, in incubation, often
The culture solution of replacement in 2-3 days.
Compared with prior art, the invention has the benefit that
(1) the present invention provides a kind of Green culture solution of optimization, keratinocyte and skin histology are used to prepare
Cultivating process in, for both preparation provide it is good growth and differentiation demand.
(2) the present invention provides keratinocyte cultural methods, by the isolated keratinocyte differentiation of skin histology
It forms, secondary culture can be carried out to the keratinocyte that culture obtains according to actual needs.
(3) fibroblastic cultural method provided by the invention, it is isolated by skin dermis, it can be according to reality
Demand carries out secondary culture to the fibroblast that culture obtains.
(4) construction method of skin histology provided by the invention, keratinocyte and fibroblast are put into three-dimensional flow
It is obtained by culture skin histology in formula bioreactor, can be used for Epidermic Grafting.
(5) production cost of skin histology provided by the invention is low, and biocompatibility is good, and no rejection is greatly reduced
The cost of Epidermic Grafting.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the electron microscope of cell and bracket during dimensional culture in the embodiment of the present invention 2；
Fig. 2 is the outside drawing for the skin histology cultivated in the embodiment of the present invention 2；
Fig. 3 is the electron microscope for the skin histology cultivated in the embodiment of the present invention 2.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
A kind of keratinocyte and fibroblast, are prepared using following methods:
Configure following culture medium:
The Green culture solution of optimization: DMEM culture medium and Ham ' s F12 nutrient solution are 3:1 mixing by volume, then with
Following components is added in the mixed stereometer of the two: the FCS that percentage by volume is 10% is added, EGF 10ng/ml, hydrogenation can
Pine 0.4 μ g/ml, adenine 0.18mM, 5 μ g/ml of insulin, transferrins 5 μ g/ml, glutamine 2mM, iodine thyroid gland is former
0.2 μM of propylhomoserin, amphotericin B 0.625 μ g/ml, penicillin 100U/ml, 100 μ g/ml of streptomysin.
1. skin biopsy samples
Under 1.1 aseptic conditions, medial thigh skin tissue 1.5cm is taken2Size is placed in containing 10% newborn calf serum
(FBS) and in the DMEM of dual anti-(100IU/ml penicillin 100ug/ml streptomysin), 4 DEG C of preservations.
1.2 skin histologies are cut into three pieces of 1x5mm sizes, with contain 50U/ml penicillin, 50g/ml streptomysin and 50g/ml
Cleaning 10 times in the phosphate buffered saline (PBS) of gentamicin.
1.3, which are immersed in 15ml, contains dual anti-dose of 3ml (100U/ml penicillin and 100 μ g/ml streptomysins) and 4ml tryptose
In the PBS mixed liquor of enzymic digestion liquid, is kept for 4 DEG C of temperature, stood overnight.
1.4 pour into postdigestive skin in culture dish, are separated epidermis and corium with ophthalmology tweezers.
2. keratinocyte culture
2.1, by isolated epidermis, are cleaned 3 times with PBS, are then placed in conical pipe, and 4ml tryptic digestive juice is added
(0.25% pancreatin and 0.02%EDTA mixed liquor), 37 DEG C, 5%CO2, 30min is cultivated, timing is light to shake；
Digestion is terminated with the culture solution of 20% calf serum, is blown and beaten repeatedly with suction pipe to obtain single cell suspension, crosses 200 mesh
Sieve collects filtrate to remove residual residue, is centrifuged 200g 5 minutes, removes digestive juice, is cleaned 3 times, obtained with PBS
Keratinocyte cell.
2.2 suction keratinocyte cell gently is resuspended in the Green culture solution of 5ml optimization, 37 DEG C, 5%
CO2.The keratinocyte cell 8x10 obtained5It is a, etc. numbers ratio be added auxiliary 8x105A 3T3 cell.
Fresh medium KSFM is replaced after 2.3 48 hours, and EGF 10ng/ml is added.
2.4 every 3 days replacement culture solutions, PBS cleaning.
2.5 cell culture 10-15 days, cell converges up to 70-90%, obtains keratinocyte, can diluting cells, after
Continuous secondary culture, can also be frozen at once in -150 DEG C of liquid nitrogen storage tanks.
3. dermal layer of the skin culture
3.1 autologous skin fibroblast cell primary cultures
3.1.1 after separating dermal tissue and epidermal tissue, dermal tissue is cleaned 3 times with PBS, uses new aseptic operation knife
Corium is cut into the fragment of very little, fragment is transferred to containing dual anti-dose of 0.5ml and 10ml clostridiopetidase A II type (200IU/ml)
In 50ml centrifuge tube.
3.1.3 it places, 37 DEG C, 2 hours, it is appropriate to shake.
3.1.4 2ml PBS is added, blows and beats repeatedly, then by 70 μm of cell filters, it is unicellular to obtain fibroblast
Suspension.
3.1.5 single cell suspension, 500G are centrifuged 5min, abandon supernatant, are resuspended in 2ml containing 10% fetal calf serum and 0.4 μ g/
The DMEM culture solution of ml hydrocortisone.
3.1.6 with 2x105cells/cm2Density inoculating cell.
3.1.7 in 37 DEG C and 5%CO2Lower culture cell, cultivates fluid exchange for the first time after 24-48 hours, and then every 2-3 days
Replacement is primary.
3.2 fibroblast secondary cultures
3.2.1 converge when fibroblast reaches 90%, remove culture solution, 2ml PBS is cleaned 3 times, and a few minutes, which move back, removes
PBS is added 1ml tryptic digestive juice, 37 DEG C, cultivates 3-5 minutes.
3.2.2 digestion is terminated with the culture solution of 20% calf serum, is blown and beaten repeatedly with suction pipe to obtain single cell suspension,
225G be centrifuged 5min, abandon supernatant, kind in 2ml contain 10% fetal calf serum, the DMEM culture solution of 0.4 μ g/ml hydrocortisone,
1000cell/cm2。
3.2.3 new culture dish, cell line, time and passage number are marked, in 37 DEG C and 5%CO2Lower culture cell, 24-
Fluid exchange is cultivated after 48 hours for the first time, then replacement in every 2-3 days is primary.
3.2.4 90% converge after, obtained fibroblast is spare, or is frozen in -150 DEG C of liquid nitrogen storage tanks at once.
Embodiment 2
A kind of construction method of skin histology, comprising the following steps:
Configure following culture medium:
The Green culture solution of optimization: DMEM culture medium and Ham ' s F12 nutrient solution are 3:1 mixing by volume, then with
Following components is added in the mixed stereometer of the two: the FCS that percentage by volume is 10% is added, EGF 10ng/ml, hydrogenation can
Pine 0.4 μ g/ml, adenine 0.18mM, 5 μ g/ml of insulin, transferrins 5 μ g/ml, glutamine 2mM, iodine thyroid gland is former
0.2 μM of propylhomoserin, amphotericin B 0.625 μ g/ml, penicillin 100U/ml, 100 μ g/ml of streptomysin.
Using fibroblast made from embodiment 1 and keratinocyte.
Fibroblast kind in bracket, density 5x105/cm2, the interior Green culture solution that 1ml optimization is added of bracket.
2ml RPMI1640 culture solution is added in branch frame peripheral.
37 DEG C, 5%CO2Culture 3 days, changes 1 culture solution in every 2 days.
4th day, in rack surface kind keratinocyte (1x106), cells/ml it is adhered to after 24 hours.
At 3-5 days, gas-liquid interface is formed.Specifically as shown in Figure 1.In Fig. 1, Figure 1A is 800 times of scanning electron microscope
Figure, Figure 1B are 3000 times of scanning electron microscope diagrams, and Fig. 1 C is 3000 times of scanning electron microscope diagrams, and Fig. 1 D is 6000 times of scannings
Electron microscope picture, Fig. 1 E are 800 times of scanning electron microscope diagrams, and Fig. 1 F is 1600 times of scanning electron microscope diagrams, Tu1BHe
K indicates keratinocyte or fibroblast in 1F, and F indicates bracket.It can be seen that fibroblast and angle from Figure 1A and 1B
Matter formation cell is orderly very well to be adhered on three dimensional scaffold structure；Fig. 1 C and 1D can be seen that horn cell and at fiber finer
Born of the same parents are attached on three dimensional scaffold structure well, and the stimulating growth that interacts, and on bracket upper layer, form keratinocyte layer；Figure
1E and 1F can be seen that every kind of cell all well and bracket peripheral cell merge into each other promotion.
37 DEG C, 5%CO2, continue to cultivate, change within every 3 days 1 culture solution, self-forming gas-liquid interface calculates, and continues culture 21
The skin histology of portable can be obtained in it.
The outside drawing of obtained skin histology is as shown in Fig. 2, electron microscope is as shown in Figure 3.
From figure 3, it can be seen that skin histology produced by the present invention has the structure of normal tissue, meet related skin through detection
The regulation of skin tissue.
Wherein, equipment is the three-dimensional streaming bioreactor of Mai Dekesi Biotechnology Co., Ltd of Germany.
A kind of construction method of skin histology provided by the invention, obtained skin histology be people's autologous skin tissue etc.
Object is imitated, all has good skin properties and stable biochemical characteristic, and since raw material is derived from human body itself, is had good
Biocompatibility, no rejection, and cost is relatively low needed for entire production process.
Through lot of experiment validation, skin histology provided by the invention is successfully applied to skin graft operation.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of Green culture solution of optimization, which is characterized in that DMEM culture medium and Ham ' s F12 nutrient solution are 3 by volume
Following components is added in the mixed liquor of ± 0.2:1 composition: in terms of the volume of the mixed liquor, 10 ± 1ng/ml of EGF, hydrogenation can
Pine 0.4 ± 0.05 μ g/ml, 0.18 ± 0.02mM of adenine, 5 ± 0.5 μ g/ml of insulin, 5 ± 0.5 μ g/ml of transferrins,
2 ± 0.2mM of glutamine, 0.2 ± 0.05 μM of iodine thyronine, FCS and antibiotic, the volume hundred of the FCS addition
Score is the 10% ± 1% of the mixeding liquid volume.
2. the Green culture solution of optimization according to claim 1, which is characterized in that the antibiotic includes anphotericin
B, penicillin, streptomysin are any one or more of；
Further, in terms of the volume of the mixed liquor, the additive amount of each antibiotic are as follows: 0.625 ± 0.05 μ g/ of amphotericin B
Ml, 100 ± 5U/ml of penicillin, 100 ± 5 μ g/ml of streptomysin.
3. a kind of keratinocyte cultural method, which comprises the following steps:
Keratinocyte is resuspended in the Green culture solution of the described in any item optimizations of claim 1-2, and auxiliary 3T3 cell is added,
Culture；
KSFM culture medium is replaced after 7-8 hours, and 10 ± 1ng/ml of EGF is added；
Every 3 days replacement culture solutions, PBS cleaning, culture obtain the keratinocyte.
4. keratinocyte cultural method according to claim 3, which is characterized in that the keratinocyte with it is described
The number ratio of 3T3 cell is 1:0.8-1.2；
Further, the keratinocyte is digested isolated by epidermal tissue；
Further, the epidermal tissue is isolated by thigh skin, preferably medial thigh skin；
Further, the time of the culture is 10-15 days, and cell converges up to 70%-90%, obtains the cutin and is formed carefully
Born of the same parents；
Further, after the cell converges up to 70%-90%, further includes: diluting cells continue secondary culture, obtain described
Keratinocyte.
5. a kind of fibroblastic cultural method, which comprises the following steps:
Dermal tissue uses collagenase digesting, is isolated to fibroblast single cell suspension；
The fibroblast single cell suspension is added in the DMEM culture solution containing fetal calf serum and hydrocortisone, with institute
The stereometer of DMEM culture solution is stated, the addition volume of the fetal calf serum is 10% ± 1%, 0.4 μ g/ of the hydrocortisone
ml；
Culture, cultivates fluid exchange after 24-48 hours, then the culture solution of replacement in every 2-3 days for the first time, until 85%-90% converges
After conjunction, the fibroblast is obtained.
6. fibroblastic cultural method according to claim 5, which is characterized in that the culture are as follows: at 37 ± 2 DEG C
And 5%CO2Lower culture cell；
Further, the dermal tissue is isolated by thigh skin, preferably medial thigh skin；
Further, the dermal tissue and epidermal tissue as claimed in claim 4 derive from same skin；
Further, the fibroblast obtained further includes the steps that secondary culture.
7. a kind of construction method of skin histology, which comprises the following steps:
Fibroblast kind is added described in any one of claim 1-2 in bracket in the bracket of three-dimensional streaming bioreactor
Optimization Green culture solution；
RPMI1640 culture solution is added in branch frame peripheral, cultivates 90-110h, keratinocyte is added in the rack surface,
Culture；
Gas-liquid interface is formed, culture solution is regularly replaced, continues culture 21 ± 1 days.
8. the construction method of skin histology according to claim 7, which is characterized in that the fibroblast is in the branch
Density in frame is 5 × 105/cm2；
Further, the fibroblast uses fibroblast described in claim 5 or 6.
9. the construction method of skin histology according to claim 8, which is characterized in that the addition of the keratinocyte
Amount is 1 × 106cells/ml；
Further, the keratinocyte is using keratinocyte described in claim 3 or 4.
10. the construction method of skin histology according to claim 8, which is characterized in that in the construction method of skin histology
The condition of culture is 37 ± 1 DEG C, 5%CO2, in incubation, the culture solution of replacement in every 2-3 days.
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| CN113528426A (en) * | 2021-05-27 | 2021-10-22 | 江苏省人民医院（南京医科大学第一附属医院） | Method for culturing fibroblast from autologous skin |
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| CN113528426A (en) * | 2021-05-27 | 2021-10-22 | 江苏省人民医院（南京医科大学第一附属医院） | Method for culturing fibroblast from autologous skin |
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