Method of autologous primary hair follicles preparation in 3D culture
The present invention relates to a method for the preparation of autologous human primary hair follicles in 3D cultures, comprising the isolation of primary fetal follicles; isolation of the patient's hair follicle cells; isolation of skin cells of the patient's scalp; extraction of growth factors from fetal follicle cells; the fibrin gel creation that contains growth factors of fetal follicles; sandwich cultivation of patient's hair follicle cells and skin of the patients scalp on or into fibrin gel that contains growth factors of fetal follicles; separation from fibrin gel the patients primary hair follicles, which can be used to treat baldness as an autologous graft.
1. A method for creating de novo autologous primary hair follicles in 3D culture by recreating the conditions of embryogenesis of primary hair follicles, which involves: isolating mesenchymal stem cells of donor hair follicles and their expansion; isolating of donor scalp skin cells and their temporary cryopreservation; seeding, using a sandwich method, mesenchymal stem cells of the donor's hair follicles and skin cells of the donor's scalp in a monolayer of fibrin gel based on a nutrient medium containing growth factors of fetal scalp hair follicles; lysing of the fibrin gel monolayer with exogenous plasmin; isolating de novo-formed primary donor hair follicles from a lysed fibrin gel monolayer by self-precipitation; washing the de novo-formed primary hair follicles of the donor with phosphate buffer; transplanting of de novo-formed primary hair follicles to a donor.
2. The method for creating de novo autologous primary hair follicles according to claim 1 , wherein:
(i) extraction of hair follicles from the donor's scalp is performed by simultaneously collecting two follicular units with small (1-2 mm 3 ) pieces of donor scalp skin located between them;
(ii) isolation of mesenchymal stem cells of the donor's hair follicles and their expansion in 2D culture is carried out using standard procedures and standard reagents by separation of adhesive mesenchymal cells of the donor's scalp hair follicles after achieving 70-80% confluency;
(iii) the method of preparation of autologous primary hair follicles according to (i), wherein isolation of donor scalp skin cells is carried out by soft homogenization of the donor scalp skin located between the extracted hair follicles in order to create a cell suspension containing interfollicular stem/progenitor cells of the donors' skin;
(iv) the method of preparation of autologous primary hair follicles according to (iii), wherein temporary preservation of donor scalp skin cells suspension that contains interfollicular stem/progenitor cells of the donors' skin is performed by programmable cryopreservation in liquid nitrogen according to a standard procedure with the aim of temporarily preserving this cell suspension for 2-3 weeks—the period which necessary for the expansion of mesenchymal stem cells of the donor's scalp hair follicles; and defrosting of a suspension of skin cells from the donor's scalp is carried out according to a standard procedure.
3. The method for creating de novo autologous primary hair follicles according to claim 1 , wherein seeding using a standard sandwich method of mesenchymal stem cells of the donor's scalp hair follicles and a suspension of donor scalp skin cells for 3D cultivation is carried out in a monolayer of fibrin gel based on a nutrient medium containing growth factors of the fetal scalp skin hair follicles of a fetus of 18-20 weeks of gestation obtained as a result of medical termination pregnancy independent of the researcher.
4. The method for creating de novo of autologous primary hair follicles according to claim 3 , wherein fetal growth factors are extracted by gently homogenizing the primary hair follicles of the fetal scalp, and followed centrifugation and filtration of the resulting supernatant.
5. The method for creating de novo autologous primary hair follicles according to claim 3 , wherein the growth factors and concentrations of the growth factors of the fetal scalp skin hair follicles: HGF-43.91±11.84 pg/ml; IGF-1-432.6±65.76 pg/ml; VEGF-26.55±10.43 pg/ml; FGF-7-121.90±37.10 pg/ml; EGF-2.82±0.92 pg/ml; SCF-440.70±19.52 pg/ml; TGFβ1-82.13±1.38 pg/ml; TNFα-249.53±10.40 pg/ml; ANGPT1-12970.00±1062.39 pg/ml; bFGF-9540.00±674.25 pg/ml; VEGF-A-5360.00±381.13 pg/ml.
6. The method for creating de novo autologous primary hair follicles according to claim 4 , wherein biosafety control of fetal scalp primary hair follicles growth factors is carried out according to generally accepted guidelines developed for regenerative medicine.
7. The method for creating de novo autologous primary hair follicles according to claim 1 , wherein lysis of the fibrin gel monolayer after de novo formation primary hair follicles is performed by exogenous plasmin at a concentration not less then 1.0 mg/ml.
8. The method for creating de novo autologous primary hair follicles according to claim 1 , wherein isolation of de novo primary donor's hair follicles from a lysed fibrin gel monolayer is performed by self-precipitation.
9. The method for creating de novo autologous primary hair follicles according to claim 1 , wherein washing of the de novo formed primary hair follicles of the donor is carried out three times with phosphate buffer in order to clean the primary hair follicles from impurities of plasmin, fibrin-degradation products and the cultivation medium.
10. The method for creating de novo autologous primary hair follicles according to claim 1 , wherein transplantation of de novo-formed primary hair follicles to a donor is carried out according to a standard method in cases where the hair follicles on the bald skin of the donor's scalp are dead and are not subject to stimulating growth such as chemical burns, toxic, hormonal and other factors that lead to persistent alopecia due to complete destruction of hair follicles.