CN1834235A - Method of fast separating epithelial cell from human scalp hair follicle - Google Patents

Abstract

This invention discloses a high-performance rapid separation method for hair dermal papilla cells, which comprises the steps of: using collagenase I to digest scalp, then using pancreatin to digest scalp to obtain hair dermal papilla cell clones, inoculating and culturing. The method can treat scalp in a large scale and separate hair dermal papilla cells, thus having such advantages as high performance and rapid acting.

Description

Epithelial method in a kind of efficient sharp separation number of people fur capsule

Technical field:

The present invention relates to the separation of cell, be specifically related to epithelial separation in the number of people fur capsule.

Background technology:

The survey showed that to 6000 25 years old-49 years old male sex according to Beijing Hong Hui biopharmaceutical company in 2003, also increasing with the sickness rate of the increase androgenetic alopecia at age.Wherein, the sickness rate between 25 years old-29 years old is that the sickness rate between 12%, 45 years old-49 years old is 31%.Modern medicine study shows that the epithelial cell in the hair follicle can be used to make up engineered hair follicle, and engineered hair follicle can be used for hair transplantation.But, make up engineered hair follicle, just need the epithelial cell of a large amount of hair follicles.

Separating epithelial method at present from hair follicle has following several:

1. micro-partition method

Expose with trysinization to external root sheath at the microscopically branch, then single hair follicle is inoculated in the culture dish and (, accords with just Yang Tian referring to height power; The vitro culture of outer root sheath cells of murine vibrissa follicles and uPA and uPAR expression study; Third Military Medical University's journal; 2004; 26 (16): 1452).This method is owing to will carry out the isolating operation of single hair follicle at microscopically, and velocity of separation is slow, and labour intensity is big, and cell easily pollutes.

2. dep

From scalp, directly extract hair follicle, after trysinization becomes individual cells, cultivate.Perhaps scalp is extracted hair follicle after collagenase and the digestion of pancreatin mixed solution, put and be digested to the individual cells cultivation in the pancreatin (referring to Sotaro K, Satoshi I, IIirotoT.Successful transplantation of cultural human outer root sheath cells asepithelium.Annals Plastic Surgery, 1994,33 (3): 290).This method also will be carried out single hair follicle operation at microscopically, also has the slow deficiency of velocity of separation.State knows and office disclosed the application for a patent for invention of a kind of " adhesion method separation and concentration hair follicle stem cells fast " in 2005 03 year 16 days (publication number is: CN1594554), the disclosed method of this patent application also belongs to this dep, still has the deficiency of described dep equally.

3. digestion extrusion molding

The scalp bar is after corium subcutis intersection is cut off, separate enzyme with 0.5% and digest 16-18h down at 4 ℃, epidermis is separated with corium, from straight skin and subcutis, extrude the follicular epithelium composition then (referring to Wu Jinjin, Liu Rongqing, leaf celebrating a dance squad etc. the proliferative ability of each section of people follicular epithelium cell cultures is relatively; Third Military Medical University's journal; 1999; 21 (11): 788).This method is oversize to the scalp digestion time, and the follicular epithelium composition of extruding is imperfect, not easily collecting.

This shows that the separation method of follicular epithelium cell described in the prior art all is single hair follicle separation method, not only labour intensity is big, and velocity of separation is also slow, so epithelial cell is the technical barrier that solution is thirsted in this area in the sharp separation number of people fur capsule.

Summary of the invention

Technical problem to be solved by this invention is an epithelial sharp separation in the number of people fur capsule.The present invention has monoblock batch processing, efficient advantage fast.

The technical scheme that the present invention solves the problems of the technologies described above may further comprise the steps successively:

A. the processing of people's scalp: remove fat and reticular tissue in outer hair of scalp and the subcutis, make the wide leather strap of 0.3-0.5cm, 10-15min soaks with the liquor hibitane of 2.5g/L in elder generation, washs three times with the phosphate buffered saline buffer concussion of 0.1mol/L again;

B. enzyme digestion digestion: remove epidermis, broken, under 37 ℃, stir and add 0.5% collagenase I with 100rpm speed, stir after 2 hours suspension and cross 800 mesh sieves, be retained in hair shaft on the screen cloth with the phosphate buffered saline buffer filtration washing of 0.1mol/L again, collect the back and add 0.075% pancreatin down at 25 ℃ and left standstill 20 minutes;

C. culture medium preparation: add foetal calf serum, Regular Insulin, hydrocortisone and gentamicin at the K-SFM substratum, make in every milliliter of K-SFM substratum and contain: 0.1ml foetal calf serum, 6ug Regular Insulin, 0.5ug hydrocortisone and 40ug gentamicin;

D. the substratum of c step gained is added in the solution of b step gained, with 1000rmp speed centrifugal 3 times, each centrifugal 5 minutes, outwell supernatant liquor, obtain the epithelial cell group;

E. in incubator, overlay concentration and be 1% type i collagen, the resulting epithelial cell group of steps d is inoculated into cultivates in the incubator just again.

People's scalp of the present invention maybe needs to carry out the patient's of plastic surgery operations scalp for carrying out hair transplantation.

Incubator of the present invention is that specification is the culture dish of 35mm.

Epithelial method is carried out batch processing to the patient from body monoblock scalp in the efficient sharp separation number of people fur capsule of the present invention, obtains the epithelial cell in the hair follicle, has that labour intensity is low imitates fireballing unusual effect with separating.Adopt described method to separate the transplanting that the epithelial cell that obtains can be used for hair.

Embodiment:

Below be embodiments more of the present invention and effect, but these embodiment do not do any type of restriction to the present invention.

Embodiment one:

1. shave except that patient's occipitalia hair, mark cuts scope, 2 * 6cm, and medical alcohol sterilization 3 times is cut along mark line with 11 trumpeter's art blades, reaches fascia layer deeply, takes off scalp, occipitalia otch 3-0 silk suture;

2. fat and the reticular tissue in outer hair of removal scalp and the subcutis made the wide leather strap of 0.3-0.5cm, and the liquor hibitane with 2.5g/L soaks 15min (solution covers leather strap) earlier, and the phosphate buffered saline buffer with 0.1mol/L shakes washing three times again;

3. remove the epidermis of the resultant scalp of step 2, broken, under 37 ℃, stir and add 0.5% collagenase I with 100rpm speed, stir after 2 hours suspension and cross 800 mesh sieves, phosphate buffered saline buffer filtration, washing with 0.1mol/L is retained in the hair shaft on the screen cloth again, and the pancreatin of collecting back adding 0.075% under 25 ℃ left standstill 20 minutes;

4. add foetal calf serum, Regular Insulin, hydrocortisone and gentamicin at the K-SFM substratum, make in every milliliter of K-SFM substratum and contain: 0.1ml foetal calf serum, 6ug Regular Insulin, 0.5ug hydrocortisone and 40ug gentamicin;

5. the substratum with 4 step gained adds in the solution of 3 step gained, and with 1000rmp speed centrifugal 3 times, each centrifugal 5 minutes, outwell supernatant liquor, obtain the epithelial cell group;

6. in the culture dish of 35mm, overlay concentration and be 1% type i collagen, the resulting epithelial cell group of step 5 is inoculated in the culture dish cultivates again.

Used 0.5% collagenase I, 0.075% pancreatin, type i collagen are all available from Sigma company;

Used K-SFM substratum is purchased the company in Gibco, foetal calf serum is purchased in Hangzhou folium ilicis chinensis company, Regular Insulin is purchased the pharmacy in Xuzhou confederate state, hydrocortisone is purchased in Yangzhou, Jiangsu pharmaceutical factory, gentamicin is purchased in Xuzhou drugmaker of confederate state, liquor hibitane is purchased in North China pharmaceutical Co. Ltd, and the phosphate buffered saline buffer of 0.1mol/L is an autoclaved phosphate buffered saline buffer of purchasing Yu Weijia company

Embodiment two

1. press the step 1 of embodiment one, obtain the scalp of 1 * 3cm size;

2. fat and the reticular tissue in outer hair of removal scalp and the subcutis made the wide leather strap of 0.3-0.5cm, and the liquor hibitane with 2.5g/L soaks 10min (solution covers leather strap) earlier, and the phosphate buffered saline buffer with 0.1mol/L shakes washing three times again;

3. remove the epidermis of the resultant scalp of step 2, broken, under 37 ℃, stir and add 0.5% collagenase I (solution covers leather strap) with 100rpm speed, stir after 2 hours suspension and cross 800 mesh sieves, phosphate buffered saline buffer filtration, washing with 0.1mol/L is retained in the hair shaft on the screen cloth again, and the pancreatin (solution covers tissue) of collecting back adding 0.075% under 25 ℃ left standstill 20 minutes;

4. add foetal calf serum, Regular Insulin, hydrocortisone and gentamicin at the K-SFM substratum, make in every milliliter of K-SFM substratum and contain: 0.1ml foetal calf serum, 6ug Regular Insulin, 0.5ug hydrocortisone and 40ug gentamicin;

5. the substratum with 4 step gained adds in the solution of 3 step gained, and with 1000rmp speed centrifugal 3 times, each centrifugal 5 minutes, outwell supernatant liquor, obtain the epithelial cell group;

6. in the culture dish of 35mm, overlay concentration and be 1% type i collagen, the resulting epithelial cell group of step 5 is inoculated in the culture dish cultivates again.

Embodiment three

1. repeat the step 1 of embodiment one, get the scalp of 1.5 * 4cm size;

2. the processing of people's scalp: remove fat and reticular tissue in outer hair of scalp and the subcutis, make the wide leather strap of 0.3-0.5cm, liquor hibitane with 2.5g/L soaks 12min (solution covers leather strap) earlier, washs three times with the phosphate buffered saline buffer concussion of 0.1mol/L again;

3. enzyme digestion digestion: the epidermis of removing the resultant scalp of step 2, broken, under 37 ℃, stir and add 0.5% collagenase I (solution covers leather strap) with 100rpm speed, stir after 2 hours suspension and cross 800 mesh sieves, phosphate buffered saline buffer filtration, washing with 0.1mol/L is retained in the hair shaft on the screen cloth again, and the pancreatin (solution covers tissue mass) of collecting back adding 0.075% under 25 ℃ left standstill 20 minutes;

4. culture medium preparation: add foetal calf serum, Regular Insulin, hydrocortisone and gentamicin at the K-SFM substratum, make in every milliliter of K-SFM substratum and contain: 0.1ml foetal calf serum, 6ug Regular Insulin, 0.5ug hydrocortisone and 40ug gentamicin;

5. the substratum with 4 step gained adds in the solution of 3 step gained, and with 1000rmp speed centrifugal 3 times, each centrifugal 5 minutes, outwell supernatant liquor, obtain the epithelial cell group;

6. in the culture dish of 35mm, overlay concentration and be 1% type i collagen, the resulting epithelial cell group of step 5 is inoculated in the culture dish cultivates again.

The cultivation of embodiment four follicular epithelium cells

Get embodiment one, two or three resulting epithelial nutrient solutions by the known method cultivation of going down to posterity, get the 3rd generation cell, outwell nutrient solution, add concentration and be 0.1% pancreatin 1ml, control digestion under inverted microscope, discard pancreatin after the cell shrinkage, add the substratum 2ml that presses the preparation of embodiment one step 4, dispel cell, adjust cell concn to 1 * 105/ml, be inoculated in 24 well culture plates, divide 8 groups, every group of 3 holes.Cultivate and to obtain a large amount of required epithelial cells of structure hair follicle tissue after 8 days.

Claims (1)

1. epithelial method in the efficient sharp separation number of people fur capsule may further comprise the steps successively:

A. the processing of people's scalp: remove fat and reticular tissue in outer hair of scalp and the subcutis, make the wide leather strap of 0.3-0.5cm, 10-15min soaks with the liquor hibitane of 2.5g/L in elder generation, washs three times with the phosphate buffered saline buffer concussion of 0.1mol/L again;

B. enzyme digestion digestion: remove epidermis, broken, under 37 ℃, stir and add 0.5% collagenase I with 100rpm speed, stir after 2 hours suspension and cross 800 mesh sieves, be retained in hair shaft on the screen cloth with the phosphate buffered saline buffer filtration washing of 0.1mol/L again, collect the back and add 0.075% pancreatin down at 25 ℃ and left standstill 20 minutes;

C. culture medium preparation: add foetal calf serum, Regular Insulin, hydrocortisone and gentamicin at the K-SFM substratum, make in every milliliter of K-SFM substratum and contain: 0.1ml foetal calf serum, 6ug Regular Insulin, 0.5ug hydrocortisone and 40ug gentamicin;

D. the substratum of c step gained is added in the solution of b step gained, with 1000rmp speed centrifugal 3 times, each centrifugal 5 minutes, outwell supernatant liquor, obtain the epithelial cell group;

E. in incubator, overlay concentration and be 1% type i collagen, the resulting epithelial cell group of steps d is inoculated into cultivates in the incubator just again.