CN103497930A - Dermal papilla cell culture method - Google Patents
Dermal papilla cell culture method Download PDFInfo
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- CN103497930A CN103497930A CN201310451331.2A CN201310451331A CN103497930A CN 103497930 A CN103497930 A CN 103497930A CN 201310451331 A CN201310451331 A CN 201310451331A CN 103497930 A CN103497930 A CN 103497930A
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Abstract
The invention discloses a dermal papilla (DP) cell culture method. The dermal papilla cell culture method can be used for culturing DP cells for a long term and is successfully applied to DP cell line establishment.
Description
Technical field
The invention belongs to biological technical field, in particular to a kind of cultural method of hair papilla cell.
Background technology
Hair has corresponding density and form at the skin privileged site, and it has outside defence, defencive function human body, also has the information interchange of increasing and effect attractive in appearance.Hair is generated by hair follicle, and hair follicle has the characteristic of periodic regeneration, experiences vegetative period, catagen, the circulation of stationary phase.Wherein, by the conversion in stationary phase to vegetative period, starting vegetative period is the link of most critical.When the periodic regeneration function generation obstacle of hair follicle, just there will be the hair follicle relative diseases such as alopecia, alopecia areata.Along with social development, people more and more pay attention to the hair of beauty function having protection, to the research of hair follicle periodic regeneration, will seem more and more important.
Because hair follicle has the periodically characteristic of growth, for tissue regeneration research provides an extraordinary model.The periodicity growth of hair follicle is occurred by the hair follicle stem cells mediation, the same with other stem cells, and the tranquillization of hair follicle stem cells and activation are subject to the regulation and control of stem cell microenvironment.Hair follicle stem cells is positioned at the Hair Follicle Bulge district, and it is also few that people understand the microenvironment factor that affects hair follicle stem cells self and differentiation.
Attractive especially, in the cyclic activity process of hair follicle stem cells, hair papilla (dermal papilla, DP) occupy very special status.DP is the cell in the mesenchyme source of a group specialization in corium.Large quantity research shows: postnatal each hair follicle in the cycle DP and hair follicle stem cells all can occur regular, regular contact, and this contact is the tightst when starting in the static end of term or vegetative period, illustrate that the DP cell may provide the microenvironment support for hair follicle stem cells, and play a role at the initial period of hair follicle periodic regeneration.Gene knockout research discovery, the DP disappearance, can not form complete hair follicle 2; Find again subsequently, DP is the center of an excreted factor, can generate a large amount of signaling molecules; Nearest research shows, the signal path that these signaling molecules activate is the important regulating and controlling factor that hair follicle is periodically grown, and they are bringing into play different regulating effects at the different times of hair follicle growth.
Have been reported, the DP cell is made to micro-capsule and can induce the rat ears hair follicle regeneration, and the clinical trial report for the alopecia areata treatment by people DP cell conditioned medium is arranged.These application all need a large amount of DP cells, and therefore, a large amount of cultivations of DP cell are one of hair follicle Periodic correlation disease treatment gordian techniquies that need to solve.Many scholars attempt carrying out the former culture of DP cell, find the increase along with DP cell cultures algebraically, and its biologic activity changes fast; When being passaged to for the 6th generation, cell is lost the characteristic of inducing hair follicle growth.
Visible, the DP cell has extremely important effect at hair follicle in the cycle.Therefore, obtain the essential condition that a large amount of DP cells just becomes treatment hair follicle cycle obstacle relative disease.
Summary of the invention
The cultural method that the purpose of this invention is to provide a kind of hair papilla (DP) cell, this culture system can long-term cultivation DP cell, and is successfully applied to the DP cell and builds and be.
One aspect of the present invention relates to a kind of cultural method of hair papilla cell, it is characterized in that comprising the steps:
The preparation of obtain solution: 1.0.25%Dispase (neutral protease, also referred to as Dispase) solution: accurately take 0.25g Dispase enzyme powder, add 100ml0.01M PBS solution, suction filtration packing after dissolving fully, 4 ℃ or-20 ℃ of preservations;
2.2mg/ml the preparation of collagenase: with TESCA damping fluid (50mM TES, 0.36mM CaCl
2, pH7.4) 37 ℃ of preparations, after preparing ,-20 ℃ of packing are preserved;
3.DP the preparation of substratum: α-MEM basic medium, add 10% foetal calf serum, 100 * Sodium.alpha.-ketopropionate, 100 * non-essential amino acid, 1000 * mycillin, 10ng/ml bFGF;
Culturing process:
1. get 1 of the healthy C57 mouse of newborn 8～9d, the cervical vertebra dislocation is put to death, cotton ball soaked in alcohol sterilization antenna position; The vertical skin of eye scissors is cut rat both sides antenna skin, puts into the culture dish that D-Hank's liquid is housed; Clip Irrigation with tweezers, suck unnecessary D-Hank's liquid;
2. with No. 27 syringe needles, hair follicle is branched away together with connective tissue sheath under dissecting microscope, and transfer in another culture dish that D-Hank's substratum is housed;
3. after separating, in super clean bench, with suction pipe, D-Hank's liquid is blotted, add the Dispase that massfraction is 0.25%, 20min (10-30min);
4. after having digested, hair follicle is transferred in large culture dish, added D-Hank's liquid by Dispase dilution (on a small quantity repeatedly), under operating microscope, hair follicle is separated from dermal sheath
5. separate hair papilla under operating microscope, collect and move into centrifuge tube;
6. add 0.2% collagenase in centrifuge tube, 37 ℃ of digestion 5-6 hour;
7. digestion, after 5-6 hour, adds the DMEM substratum to stop digestion, piping and druming, centrifugal 1000rpm, 3min in centrifuge tube;
8. abandon supernatant, cell is resuspended with the DP substratum, in 6 orifice plates, cultivates, and after cultivation 4d, observes, and ordinary method goes down to posterity;
9. cell reached for the 15th generation and when above, collecting cell.
The accompanying drawing explanation
Fig. 1: the go down to posterity fluorescent microscope photo of DP cell of former culture in 15 generations of identified by immunofluorescence.
Embodiment
One, the foundation of DP cell culture system
(1) type i collagen is coated
1. prepare the HCl of 0.01mol/L, PBS, standby after autoclaving;
2. dilution collagen: type i collagen is diluted to 50 μ g/ml with the HCl of 0.01mol/L
3. coated: the six every holes of orifice plate add 2ml (final concentration 10 μ g/cm2);
4. room temperature is placed 1h
5. sop up supernatant
6.PBS clean coated plate 3 times
7. coated plate is placed in 4 ℃ standby (can preserve 1 week)
(2) preparation of common agents
1.0.25%Dispase the preparation of (neutral protease, also referred to as Dispase) solution: accurately take 0.25g Dispase enzyme powder, add 100ml0.01M PBS solution, suction filtration packing after dissolving fully, 4 ℃ or-20 ℃ of preservations.
2.2mg/ml the preparation of collagenase: with TESCA damping fluid (50mM TES, 0.36mM CaCl
2, pH7.4) 37 ℃ of preparations, after preparing ,-20 ℃ of packing are preserved
3.DP the preparation of substratum: α-MEM basic medium, add 10% foetal calf serum, 100 * Sodium.alpha.-ketopropionate, 100 * non-essential amino acid, 1000 * mycillin, 10ng/ml bFGF.
(3) experimental procedure
1. get 1 of the healthy C57 mouse of newborn 8～9d, the cervical vertebra dislocation is put to death, cotton ball soaked in alcohol sterilization antenna position.The vertical skin of eye scissors is cut rat both sides antenna skin, puts into the culture dish that D-Hank's liquid is housed.Clip skin with tweezers and slightly do cleaning, suck unnecessary D-Hank's liquid;
2. under dissecting microscope, (* 3.2) branch away hair follicle with No. 27 syringe needles together with connective tissue sheath, and transfer in another culture dish that D-Hank's substratum is housed;
3. after separating, in super clean bench, with suction pipe, D-Hank's liquid is blotted, add the Dispase that massfraction is 0.25%, 20min (10-30min);
4. after having digested, hair follicle is transferred in large culture dish, added D-Hank's liquid by Dispase dilution (on a small quantity repeatedly), under operating microscope, hair follicle is separated from dermal sheath
5. separate hair papilla under operating microscope, collect and move into centrifuge tube;
6. add 0.2% collagenase in centrifuge tube, 37 ℃ of digestion 5-6 hour;
7. digestion, after 5-6 hour, adds the DMEM substratum to stop digestion, piping and druming, centrifugal 1000rpm, 3min in centrifuge tube;
8. abandon supernatant, cell is resuspended with the DP substratum, in 6 orifice plates, cultivates, and after cultivation 4d, observes, and ordinary method goes down to posterity;
9. cell reached for the 15th generation and when above, collecting cell.
10. while being passaged to for the 15th generation, the DP cell of cultivating with the culture system of setting up still expresses DP cell specific marker thing FGF7 and a-SMA is identified, qualification result shows the DP cell culture system that (as shown in Figure 1) sets up, cultivate the DP cell after 15 generations, cell still keeps the primary cell characteristic, and experiment further confirms that it still has the characteristic of inducing hair follicle growth.Therefore, utilize the method can obtain enough DP cells, for scientific research and clinical treatment.The DP cell strain of setting up, have the gene expression pattern identical with primary DP cell.Therefore, except for scientific research and clinical treatment, also can be used for large-scale pilot scale research.
The above is the preferred embodiments of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. the cultural method of a hair papilla cell, is characterized in that comprising the steps:
Obtain solution:
The preparation of 0.25%Dispase (neutral protease, also referred to as Dispase) solution: accurately take 0.25g Dispase enzyme powder, add 100ml0.01M PBS solution, suction filtration packing after dissolving fully, 4 ℃ or-20 ℃ of preservations;
The preparation of 2mg/ml collagenase: with TESCA damping fluid (50mM TES, 0.36mM CaCl
2, pH7.4) 37 ℃ of preparations, after preparing ,-20 ℃ of packing are preserved;
The preparation of DP substratum: α-MEM basic medium, add 10% foetal calf serum, 100 * Sodium.alpha.-ketopropionate, 100 * non-essential amino acid, 1000 * mycillin, 10ng/ml bFGF;
Culturing process:
1) get 1 of the healthy C57 mouse of newborn 8～9d, the cervical vertebra dislocation is put to death, cotton ball soaked in alcohol sterilization antenna position; The vertical skin of eye scissors is cut rat both sides antenna skin, puts into the culture dish that D-Hank's liquid is housed; Clip Irrigation with tweezers, suck unnecessary D-Hank's liquid;
2) under dissecting microscope, (* 3.2) branch away hair follicle with No. 27 syringe needles together with connective tissue sheath, and transfer in another culture dish that D-Hank's substratum is housed;
3) after separating, in super clean bench, with suction pipe, D-Hank's liquid is blotted, add the Dispase that massfraction is 0.25%, room temperature digestion 10-30min, preferably 20min;
4) after having digested, hair follicle is transferred in large culture dish, added D-Hank's liquid that Dispase is diluted, under operating microscope, hair follicle is separated from dermal sheath;
5) separate hair papilla under operating microscope, collect and move into centrifuge tube;
6) add 0.2% collagenase in centrifuge tube, 37 ℃ of digestion 5-6 hour;
7) digestion, after 5-6 hour, adds the DMEM substratum to stop digestion, piping and druming, centrifugal 1000rpm, 3min in centrifuge tube;
8) abandon supernatant, cell is resuspended with the DP substratum, in 6 orifice plates, cultivates, and after cultivation 4d, observes, and ordinary method goes down to posterity;
9) cell reached for the 15th generation and when above, collecting cell.
2. the constructed hair papilla cell of cultural method claimed in claim 1 is.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2937098A1 (en) * | 2014-04-24 | 2015-10-28 | Clinica Dermatologica Ercilla | A pharmaceutical composition comprising a suspension of total cells obtained from hair follicle and plasma derived growth factors for promoting hair follicle regeneration |
| CN108359633A (en) * | 2018-04-19 | 2018-08-03 | 山东农业大学 | A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method |
| CN113621556A (en) * | 2021-08-25 | 2021-11-09 | 南方医科大学南方医院 | Construction method of androgenetic alopecia cell model |
| WO2022142046A1 (en) * | 2020-12-28 | 2022-07-07 | 上海宜治生物科技有限公司 | Human scalp hair follicle single-cell suspension, and preparation method therefor and use thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1889988A (en) * | 2003-12-05 | 2007-01-03 | 英泰格伦斯生物株式会社 | Hair growth method |
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2013
- 2013-09-29 CN CN201310451331.2A patent/CN103497930A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1889988A (en) * | 2003-12-05 | 2007-01-03 | 英泰格伦斯生物株式会社 | Hair growth method |
Non-Patent Citations (3)
| Title |
|---|
| 周洪军 等: "人毛囊外根鞘隆起、真皮鞘和毛乳头细胞高效同步分离培养的方法", 《南方医科大学学报》 * |
| 李玲: "毛乳头细胞及其部分相关生长因子的研究", 《河北医科大学硕士研究生学位论文》 * |
| 陈先才 等: "胶原酶消化法分离、培养大鼠触须毛乳头的方法", 《现代医院》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2937098A1 (en) * | 2014-04-24 | 2015-10-28 | Clinica Dermatologica Ercilla | A pharmaceutical composition comprising a suspension of total cells obtained from hair follicle and plasma derived growth factors for promoting hair follicle regeneration |
| CN108359633A (en) * | 2018-04-19 | 2018-08-03 | 山东农业大学 | A kind of beaver rabbit dermis of skin hair papilla cell isolated culture method |
| WO2022142046A1 (en) * | 2020-12-28 | 2022-07-07 | 上海宜治生物科技有限公司 | Human scalp hair follicle single-cell suspension, and preparation method therefor and use thereof |
| CN113621556A (en) * | 2021-08-25 | 2021-11-09 | 南方医科大学南方医院 | Construction method of androgenetic alopecia cell model |
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Application publication date: 20140108 |