CN107881144A - A kind of dermal fibroblast large-scale production system - Google Patents
A kind of dermal fibroblast large-scale production system Download PDFInfo
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- CN107881144A CN107881144A CN201711430115.4A CN201711430115A CN107881144A CN 107881144 A CN107881144 A CN 107881144A CN 201711430115 A CN201711430115 A CN 201711430115A CN 107881144 A CN107881144 A CN 107881144A
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Abstract
The invention discloses a kind of method produced in batches into fibroblasts of adult human dermis.This method can gather micro biopsy with effective guarantee from adult skin, extract high-quality dermal fibroblast, and mass production in a short time, obtain available for clinical cell preparation, or cell product spin-off.Present invention specifies tissue sampling program, take tissue disinfection method, primary cell embedding formula cultivation, the dermal fibroblast screening of phenotype I types, cell products quality accreditation system.
Description
Technical field
Present invention relates in general to cell engineering field, and in particular to one kind adult's auto derma is fibroblastic
Large-scale production system.
Background technology
Cell just at home and abroad progressively deploys instead of therapy at present.In clinic skin disease and trauma care field, such as skin
The fields such as the treatment of skin defect, skin anti-aging treatment, artificial skin, except the treatment of general chemical drug thinks that clinician tastes
Examination uses bioactivity medicine or preparation, including cell preparation and its spin-off.
Cell product, in terms of the materials of cell, autogenous cell immunogenicity is low, is easiest to be received by user, enters
Clinical practice.Autogenous cell materials position, materials area, speed of production, product vigor and purity, direct relation cell products
Production time and yield.Both at home and abroad by studying for many years, the method for a variety of cultivation human skin cells, but each family have been generated
Way method differs, and it is also uneven to cultivate quality.
The present invention draws materials position, operation and tissue disinfection method, primary culture method, thin to skin fibroblasts live body
Born of the same parents' phenotypic screen, cells finished product identification, production overall process quality inspection system, have carried out retrofit, the materials amount stablized-thin
Born of the same parents obtain magnitude relation, and the method for steady production.This method can obtain high-purity, high quality within the time of near steady
, the I type dermal fibroblasts of constant rate of production.
The content of the invention
One aspect of the present invention, there is provided adult's fibroblastic production method of auto derma, including living tissue disappear
Poison and transportation resources, dermal tissue embedding culture method, cell phenotype screening technique, cellular identification method.
The invention provides a kind of production method of dermal fibroblast, comprise the following steps：
(1) skin biopsies are gathered, including field of operation disinfecting process and tissue disinfection process, are disappeared in the field of operation
The disinfection way used during poison includes alcohol or Iodophor wipes the skin biopsies of collection, during the tissue disinfection
The disinfection way used comprises at least cleaning fluid elution dermal sites.
In one embodiment, the production method of above-mentioned dermal fibroblast is further comprising the steps of：
(2) primary cell separation and culture；
(3) cell phenotype screens, wherein selection I types fibroblast carries out next step operation；
(4) expand culture and collect cell.
In one embodiment of the invention, in step (1), the disinfection way that is used in the field of operation disinfecting process
Wiped including alcohol wipe and Iodophor.
In one embodiment of the invention, in step (1), the disinfection way used during the tissue disinfection is also
Including alcohol wipe epidermis and cleaning fluid elution dermal sites.
In one particular embodiment of the present invention, in step (1), the sterilization used is combined as：Field of operation passes through wine
Essence wipes and Iodophor cleaning disinfection, organizes to sterilize by way of alcohol wipe epidermis and cleaning fluid elute dermal sites.
In one particular embodiment of the present invention, in step (1), field of operation is sterilized 3 times, and tissue is sterilized 3 times.
In one embodiment of the invention, in step (3), after cell phenotype screening, Phenotypic examination, streaming inspection are carried out
Survey and methionine detection, wherein the Phenotypic examination after preferably being dyed by Ji Samu micro- sem observation form determine, the stream
Formula detection preferably by fibroblast positive indication's thing positive rate be more than 95% and negative markers' negative rate be less than it is 5% true
It is fixed.
In one embodiment of the invention, the fibroblast positive indication thing is selected from CD73, CD90 and CD105
The group formed, the fibroblast negative markers gene are selected from the group that CD31 and CD34 are formed.
In one particular embodiment of the present invention, the fibroblast positive indication thing be CD73, CD90 and
CD105, the fibroblast negative markers gene are CD31 and CD34.
In above-mentioned production method, in step (2), the method for the primitive cell culture is trained for collagen investing tissue
The method of supporting：The certain density collagen of certain thickness is overlay in culture vessel, tissue block is fully embedded in collagen, then collagen
Upper strata adds culture medium, quiescent culture.
In above-mentioned production method, in step (2), the culture medium prescription used in described primitive cell culture is
DMEM high glucose mediums, 15%FBS, 1%NEAA, 1%L- glutamine and 1% Sodium Pyruvate.
In above-mentioned production method, in step (2), wherein, the cell obtained is gathered by primary tissue, passes through disease
After poison detection and microorganism detection, determine whether the cell is still discarded available for follow-up cell primary culture.
Wherein, inspection project includes microbial safety detection (such as Bacteria Detection, fungal detection, detection of mycoplasma, interior
Mycotoxin identification) and Viral diagnosis (such as HIV, HBV, HCV, CMV, TP) detection, inspect the blood that sample is tissue supplier by random samples.Institute
There is testing result to be necessary for negative.
In above-mentioned production method, in step (4), wherein, the cell after expanding and cultivating, examined by microorganism
Survey, qualified is collected, underproof discarded.
Brief description of the drawings
Fig. 1 shows cell flow sheet.
Fig. 2 shows cell production quantity and time plot.
Fig. 3 shows not isophenic fibroblastic aspect graph (4 times of object lens optical multiplier).
Fig. 4 shows to be inoculated with after tissue cellular morphology figure (10 times of object lens optical multiplier) after different number of days.
Fig. 5 shows that primary cell embeds formula culture cell state figure (4 times of object lens optical multiplier).
Fig. 6 shows the fluidic cell figure of cell sign thing identification.
Embodiment
Embodiment
It is further illustrated by the examples that follow the present invention.There is provided embodiment to be for illustration purposes only, and should not be solved
It is interpreted as the scope or content limiting the invention in any way.
Embodiment 1：Cell production procedure
The present invention draws materials position, operation and tissue disinfection method, primary culture method, thin to skin fibroblasts live body
Born of the same parents' phenotypic screen, cells finished product identification, production overall process quality inspection system, have carried out retrofit, the materials amount stablized-thin
Born of the same parents obtain magnitude relation, and the method for steady production.This method can obtain high-purity, high quality within the time of near steady
, the I type dermal fibroblasts of constant rate of production.
As shown in figure 1, cell production mainly includes following link：
1st, operating stage：
1) operation collection living tissue, including field of operation disinfecting process and tissue disinfection process；
2) living tissue long-distance transport.
2nd, march into the arena initial stage：
1) primary tissue collection cell；
2) cell primary culture；
Wherein, the cell obtained is gathered by primary tissue, after Viral diagnosis and microorganism detection, determines the cell
Whether still discarded available for follow-up cell primary culture.
3rd, screening：
1) cell phenotype screens；
2) high-quality clone is selected；
Wherein, after cell phenotype screening, by Phenotypic examination and flow cytometer detection, qualified is chosen to be high-quality clone, no
Qualified is discarded.
4th, production period：
1) culture is expanded；
2) cell is collected；
Wherein, the cell after expanding and cultivating, by microorganism detection, qualified is collected, underproof discarded.
Cell production specifically includes：
1st, operating stage：
1) operation collection living tissue, including field of operation disinfecting process and tissue disinfection process；
Living tissue of being grown up dermal tissue collection：Collection position is collare skin, can choose collare hair line skin nearby, with
Cover scar.
Living tissue acquisition method：Adult skin tissue, pass through surgery boring operation method.Skin is adopted using surgical biopsy to punch
Device, more than diameter 1.5mm pachydermia tissue is obtained, is deep to 0.6-0.8cm.The living tissue is transferred and stored in 4 with disinfecting apparatus
DEG C closed sterile soft environment, in sterile tissue-wash solution.
2) living tissue long-distance transport：
Living tissue long-distance transport：It is that tissue transports liquid to transport key reagents.The present invention transports liquid using special tissue, is
10% antibiotic (penicillin, streptomysin), the physiological saline of 1% mycoplasma medicine.Conveying arrangement is organized, is transported for cryogenic thermostat
Defeated case.Consumable material device, it is vertical type Cryo Equipment.Whole Transporting case, upright transport, haulage time are within 72 hours.
Disinfectant program includes：Field of operation sterilizes and living tissue sterilization.
Skin histology sterilizes sterilization procedure：Operative region, with 1% medical iodophor disinfection, such as 2 times.With 75% medical wine
Essence sterilization 1 time.Drape, operation.Tissue is immediately entered with sterilization transport liquid.
Living tissue sterilizes：Living tissue terminates in transport, before production starts, carries out disinfection.Wiped using tissue-wash solution
Wipe.In this step, tissue-wash solution is key reagents.Tissue-wash solution composition：Containing 10% antibiotic, 1% anti-mycoplasma drug.
Using the cotton swab of sterilization, 75% ethanol is dipped in, expansion skin portion wipes；Using disinfecting cotton swab, tissue-wash solution is dipped in, deploys corium
And subcutaneous part, wipe back and forth；Wipe, it is such as each 3 times.Clean cleaning fluid is changed, tissue is eluted, such as 3 times.
2nd, march into the arena initial stage：
1) primary tissue collection cell；
2) cell primary culture；
Wherein, the cell obtained is gathered by primary tissue, after Viral diagnosis and microorganism detection, determines the cell
Whether still discarded available for follow-up cell primary culture.
It is prepared by fibroblast cell primary embedding culture container：Using disposable sterile culture container, collagen is carried out
Coating.Collagen is dermal tissue matrix, and the material of dermal cell secretion, and it can provide the three-dimensional ring of cell growth
Border.Because primary cell will gradually decrease in absorption of the initial stage to collagen, encapsulating layer with incubation time.Finally disappear
During change, the effect of trypsin digestion cell is not influenceed.Its residual volume, to the Secondary Culture of cell, unrestraint effect.Glue after dilution
Former albumen, uniformly spread into container, keep consistency of thickness.It must be sealed before container use, avoid water loss.
Into the cultivation of fibroblasts of adult human dermis investment：Skin histology micromanipulation method, clip dermal tissue.Obtain
The dermal sites obtained, with microdissection operating method, shred tissue；Shearing method only is used, obtains fragment of tissue.Fragment is embedded to container
In interior collagen coating buffer.Organize inoculum density, 5mm3Dermal tissue/10cm2Culture area.Original cuiture, 37 DEG C, 5%
CO2Under conditions of, static gas wave refrigerator 7-10 days.Above cultivating system is 1%-NEAA-15%FBS- DMEM in high glucose, and with transparent
The culture vessel of matter acid coating processing.
Living tissue quality inspection：
Inspect the time by random samples：Living tissue passes through primary operation, after embedding enters culture vessel, 3 to 5 days.
Inspection project：Microbial safety detection (Bacteria Detection, fungal detection, detection of mycoplasma, endotoxin detection), takes out
Sample sheet is the nutrient solution of culture 3 days to 5 days.5 virus (HIV, HBV, HCV, CMV, TP) detections, sampling observation sample carry for tissue
The blood of donor.All testing results are necessary for negative.
3rd, screening：
1) cell phenotype screens；
2) high-quality clone is selected；
Wherein, after cell phenotype screening, by Phenotypic examination and flow cytometer detection, qualified is chosen to be high-quality clone, no
Qualified is discarded.
According to Bayreuther table sexual systems, 7 kinds of phenotypes can be shown when the dermal fibroblast of people is cultivated in vitro.
Differentiate phenotypic approach, for form differential method after dyeing, on treated cover glass, cell carries out lucky Sa after growing for cell culture
Nurse dyes, and judges which group for I types cell (i.e. elongated spindle cell).
Judge the cell after phenotype, micromanipulation method will be used, the method for direct mechanical removal, only retain the thin of I types
Born of the same parents.Minimizing technology：Under the Stereo microscope visual field, using cell shovel, non-I types cell is rejected one by one.After rejecting, using containing dual anti-
Phosphate buffer rinse remaining cell.Methionine index shows I type cell purities.
I types cell purity is identified：Fibroblast mark CD73/90/105 and negative marker gene CD31/34, cell
Flow cytometer detection is carried out, positive rate need to be more than 95%, and negative rate need to be less than 5%.The methionine detection method of I type cells, methionine refer to
Mark shows I type cell purities, and testing result need to be feminine gender.By above testing result, screening obtains high-quality into human dermis into fibre
Tie up I type cell clones.(I types fibroblast is in shuttle-type ordered arrangement, is the optimal selection of Clinical practice, reaches within 5 days propagation pair
The number phase, 10 days double 4-5 times, can continuously digest more than 12 times.II types fibroblast is epithelial cell form, and nucleus is big,
Cell tight arranges, and propagation is slow；IV fibroblasts are polygon, and core is big, and propagation is slow, and arrangement is irregular.)
The quality inspection of amplification phase：
Time：Digest for the first time
Inspection project：Cell purity detects, streaming CD series.Phenotype purity detecting, methionine content.It need to be reached with beginning a project
To positive rate index.
4th, production period：
1) culture is expanded；
The scale amplification of dermal fibroblast：Cell presses 1:4 to 1:5 amplifications, amplification in about 7 days are once, final to obtain
To 2 × 107Individual cell, is shown in Fig. 2.
2) cell is collected；
Wherein, the cell after expanding and cultivating, by microorganism detection, qualified is collected, underproof discarded.
Harvest time quality inspection：
Time：Before harvest.
Inspection project：Microbial safety detection (Bacteria Detection, fungal detection, detection of mycoplasma, endotoxin detection), takes out
Sample sheet is the nutrient solution of 3 days to 5 days before harvest.All indexs need to be feminine gender, available for Clinical practice, or the life of other products
Production.
Embodiment 2：Different disinfectant programs handle the effect to cell culture success rate
Inventor compares different disinfection way and combinations thereof, specific disinfecting process and cultivation results, is shown in Table 1, always
The result tied in table 1 finds that, when field of operation is without disinfecting process, culture success ratio only has 0, and most cell is in polluting
Or the state not grown；After field of operation disinfectant program improves, i.e., using Iodophor or alcohol wipe, and organize also to do commonly to disappear
Poison, culture success ratio is up to 50% (pollution-free situation).Finally, by optimizing field of operation and tissue disinfection program, i.e. field of operation
Using alcohol+Iodophor, tissue disinfection program is eluted using alcohol wipe epidermis area+cleaning solution, finally may be such that cell culture into
Power reaches 100%.
1. different disinfectant programs of table treat tissue cultures successful rate statistics
The disinfection way of table 2. is summarized
From the summary of table 2, field of operation sterilization is the key factor for ensureing that living tissue is clean.Because field of operation is positioned at hair
Border line, hair is luxuriant, easily hides microorganism, also, sterilised liq has very big lethal effect to dermal tissue, therefore, sterilization
Work should be carried out mainly before surgery, and the disinfection way of field of operation includes alcohol wipe, wherein further may include that Iodophor wipes；
The living tissue of taking-up, because long-distance transport and place are changed, it must also be carried out disinfection before living tissue production.Epidermis position (and
Hair follicle) it is the easy parasitic position of microorganism, therefore epidermis must be wiped with medicinal alcohol；Dermal sites are not due to connecing directly
Outside is touched, can be wiped with disinfecting cotton swab and tissue-wash solution, eluted repeatedly.Therefore, for living tissue sterilization side
Formula, dermal sites are eluted including at least cleaning fluid, preferably further including alcohol wipe epidermis.
The cell production process of a certain specific batch products of embodiment 3
1st, operation obtains tissue：It is fold position after the ear of right side to take tissue location.Field of operation disinfectant program is medicinal alcohol
Wipe one time, Iodophor wipes twice.Cutting tissue amount 1mm × 5mm pachydermia.Dermal sites volume 1mm × 5mm × 0.8mm.
2nd, tissue transhipment：Living tissue is immediately placed in sterile tube, is soaked with tissue transport liquid, sterile tube is fixed upright.Tissue
Transport case is kept for 4 DEG C, is transported 1 hour, to production site.
3rd, sterilization of marching into the arena is organized：After tissue enters production site, tissue transport case is carried out disinfection, unpacked；To sterile group
Knit pipe carry out disinfection, open pipe, take out tissue.Aseptic apparatus is operated, and medicinal alcohol wiping is carried out to the epidermis position of tissue,
Totally 3 times.Tissue elution tissue-wash solution after sterilization, three times.
4th, tissue shearing：Micromanipulation clip skin portion is into small tissue blocks.
5th, prepared by culture vessel：Above tissue mass, use 10mm culture dishes.5mL 50% hyaluronic acid is subjected to bed board,
All around shake up, ensure that bed board is homogeneous.
6th, it is inoculated with：Tissue block is immersed in coating buffer, uniformly inoculation.It is with pressed disc method, sterile cover slips that tissue block is light
Gently push down, guarantee tightly attaches with culture vessel.The cell state of 7 days and 14 days is shown in Fig. 4 after inoculation.
7th, original cuiture：Add 10mL culture medium, medium component：The high sugar of DMEM, 15%FBS, 1%NEAA, 1%L-
Glutamine, 1% Sodium Pyruvate.Static gas wave refrigerator 7 days.After primary cell embedding formula culture, proliferative activity is preferably (Fig. 5).
Culture medium was drawn at the 3rd day and carries out microbial safety detection, including：Bacterium, fungi, mycoplasma, endotoxin.Detection is complete
Portion is negative, into next stage.
8th, screen：Under Stereo microscope, with steril cell curette, by the miscellaneous type cell beyond I type fibroblasts one by one
Reject.Liquid is changed, continues to cultivate.
9th, digest first：Cell covers with, and carries out digestion process and enters P1 generations.Now, original fluid is drawn, carries out microorganism
Safety detection, including：Bacterium, fungi, mycoplasma, endotoxin.To the cell under digestion, a small amount of progress colony screening is taken.It is micro-
Bio-safety detects total negative, and phenotypic screen is qualified, and mark screening is qualified, into next stage.
10th, secondary digestion：Continue to digest for second after cell culture.Now cell enters P2 generations.
11st, digest three times：Continue third time after cell culture to digest.Now cell enters P3 generations.
12nd, cell is collected：Whole cells are collected after cell culture, carry out viable count.
13rd, cell can be carried out freezing processing, or carries out parenteral solution preparation.
Improvement of the Phenotypic Selection of embodiment 4 to cell products purity
In fibroblastic production system, screening phenotype step is vital, it has been investigated that, without sieving
Choosing is directly directly cultivated by cultivating based selective, it may appear that phenotype II type fibroblasts, phenotype IV type fibroblasts, or
Two kinds of phenotypes of person coexist, and now cellular morphology is also significantly different (Fig. 3).
And compared to not screening phenotype, if increase phenotypic screen, the cellular morphology finally obtained is homogeneous, all I types into
Fibrocyte (Fig. 3).
It is incorporated by reference into
Herein cited each patent document and the complete disclosure of scientific literature are incorporated herein by reference for institute
Purposefully.
It is equivalent
The present invention can be implemented in the case where not departing from its essential characteristic with other concrete forms.Therefore, foregoing implementation
Example is considered as illustrative, rather than the limitation to invention as described herein.The scope of the present invention is by appended claims
Book rather than represented by aforementioned specification, and be intended to fall into all in the implication and scope of the equivalents of the claims
Change is included therein.
Claims (10)
1. a kind of production method of dermal fibroblast, comprises the following steps：
(1) skin biopsies are gathered, including field of operation disinfecting process and tissue disinfection process, were sterilized in the field of operation
The disinfection way used in journey includes alcohol or Iodophor wipes the skin biopsies of collection, is used during the tissue disinfection
Disinfection way comprise at least cleaning fluid elution dermal sites.
2. the method as described in claim 1, further comprising the steps of：
(2) primary cell separation and culture；
(3) cell phenotype screens, wherein selection I types fibroblast carries out next step operation；
(4) expand culture and collect cell.
3. production method as claimed in claim 1 or 2, wherein, in step (1), used in the field of operation disinfecting process
Disinfection way include alcohol wipe and Iodophor and wipe.
4. production method as claimed in claim 1 or 2, wherein, in step (1), used during the tissue disinfection
Disinfection way also includes alcohol wipe epidermis and cleaning fluid elution dermal sites.
5. production method as claimed in claim 1 or 2, wherein, in step (3), after cell phenotype screening, carry out phenotype inspection
Survey, flow cytometer detection and methionine detection, wherein the Phenotypic examination after preferably being dyed by Ji Samu micro- sem observation form it is true
It is fixed, the flow cytometer detection preferably by fibroblast positive indication's thing positive rate be more than 95% and negative markers' negative rate it is small
Determined in 5%.
6. production method as claimed in claim 5, wherein, the fibroblast positive indication thing be selected from CD73, CD90 and
The group that CD105 is formed, the fibroblast negative markers gene are selected from the group that CD31 and CD34 are formed.
7. in the production method as described in any one of claim 1 to 6, in step (2), the method for the primitive cell culture
For collagen investing tissue cultivation：The certain density collagen of certain thickness is overlay in culture vessel, tissue block is complete
It is embedded in collagen, then collagen upper strata adds culture medium, quiescent culture.
8. in the production method as described in any one of claim 1 to 6, in step (2), make in described primitive cell culture
Culture medium prescription is DMEM high glucose mediums, 15%FBS, 1%NEAA, 1%L- glutamine and 1% Sodium Pyruvate.
9. such as any one of claim 1 to 6 methods described, wherein, in step (2), wherein, gather and obtain by primary tissue
Cell, after Viral diagnosis and microorganism detection, determine the cell whether can be used for follow-up cell primary culture still
It is discarded.
10. such as any one of claim 1 to 6 methods described, wherein, in step (4), wherein, it is thin after expanding and cultivating
Born of the same parents, by microorganism detection, qualified is collected, underproof discarded.
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| CN201711430115.4A CN107881144A (en) | 2017-12-26 | 2017-12-26 | A kind of dermal fibroblast large-scale production system |
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| CN201711430115.4A CN107881144A (en) | 2017-12-26 | 2017-12-26 | A kind of dermal fibroblast large-scale production system |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110269868A (en) * | 2019-06-12 | 2019-09-24 | 江苏艾尔康生物医药科技有限公司 | A kind of construction method containing auto derma fibroblast hyaluronic acid derivatives agent |
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