The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle

Abstract

The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle as disclosed comprises the steps of: providing initial keratinocytes to be used for creating condition medium; creating and collecting condition medium from said keratinocytes; collecting hair follicle cells containing dermal papilla and dermal sheath from patients; culturing said cells as obtained from patients using condition medium collected from the prior step; injecting cultured cells in the predetermined amount and the predetermined ratio of dermal papilla cells and dermal sheath cells in the predetermined amount area characterized in that the step of injecting and collecting condition medium from said keratinocytes comprises the step of applying a solution obtained from the mixture of defined keratinocyte-serum free medium (DK-SFM) and Mesenchymal stem cell culture medium (MSC) to said initial keratinocytes, removing defined keratinocyte-serum free medium (DK-SFM), and extracting a condition medium from the said mixture and wherein the step of culturing hair follicle cells as obtained from patients comprises the step of applying condition medium extracted and MSC in the ratio of 1:1.

Description

[0001] The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle

[0002] Field of the Invention

[0003] Biotechnology in relation to the method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle

[0004] Background of the Invention

[0005] Hair loss is a common problem with multiple causes, from hormonal sensitivities to autoimmune. Many patients seek treatment because hair loss negatively impacts their self-image. Clinically proven pharmaceutical treatments for hair loss in men include topical and oral minoxidil solution or oral finasteride or combination thereof. Minoxidil and finasteride can slow hair loss to some extent and cause regrowth in many patients, but their effects are limited and last only as long as the patient is taking the drugs. Because these medications have drawbacks, some patients choose more permanent surgical treatments.

[0006] The main surgical option for hair restoration is autologous hair transplantation, in which hair follicles are harvested from non-bald areas of the scalp and transplanted into bald areas. Transplantation involves the "one-to-one" movement of hair follicles from one location on the scalp to another, and no hair is regenerated in the donor area. Therefore, treatment is limited by the amount of donor hair available.

[0007] All available medical or surgical hair growth treatments have drawbacks, and much research is being conducted to develop improvements. Follicular cell implantation is an alternative treatment that, unlike drugs, is permanent and, unlike transplants, is not limited by the amount of donor hair. The ability to grow cells in culture would provide enough cells to derive large numbers of new follicles from a small number of donor follicles.

[0008] The idea behind dermal papilla as a cell therapy to treat hair loss is not new. However, there is no clear and specific approach in culturing procedure along with the specific procedural application of the cultured cells injected to patients. There are present techniques including Follicular Unit Excision (FUE) and Follicular Unit Transplant (FUT) which have certain limitations in which hair follicles can be transferred in the amount of more than 2000-3000 hair follicle per time. The applications of both techniques (FUE and FUT) also depend on the amount of hair follicle in donor areas. The time for the actual operation can last as long as 8 hours and therefore there is a risk of using analgesia for a long period of time. A hair loss could reoccur again when the patient is getting older.

[0009] The prior disclosures include US patent No. US20090198336A1 which discloses the method to produce in vitro proto-hair for testing agents. Mature follicle or immature proto-hair can be implanted into recipient to provide hair shaft.

[0010] US20090198336A1 disclosed the use of dermal papilla, melanocyte and keratocyte cells without mentioning amount of cells for each type to produce hair shaft.

[0011] US20090198336A1 used the medium may be supplemented with substances that further support hair growth and development. For example growth factor(s), such as FGF, TGF alpha, PDGF, any molecule(s), such as Wnt, GSK-3 inhibi tors, Sonic hedgehog or Sonic hedgehog agonists, Noggin or BMP inhibitors, antibodies, iRNA, any chemical compound (s). Such as Minoxidil, any cell-conditioned-medium, Such as keratinocyte-conditioned medium to promote proto-hair development.

[0012] However, the said prior disclosure tend to be broad and vague and do not provide the specific methods for seeding, the storage, and the required medium supplement which would lead to the ease of procedure, more natural look, and more hair strength for patients.

[0013] Summary of the Invention

[0014] One of the objectives of this invention is to provide a specific approach in culturing procedure along with the specific procedural application of the cultured cells injected to patients. This includes the finding of a solution for removing limitations in relation to Follicular Unit Excision (FUE) and Follicular Unit Transplant (FUT) techniques as mentioned.

[0015] The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle comprises the steps of: providing initial keratinocytes to be used for creating condition medium; creating and collecting condition medium from said keratinocytes; collecting hair follicle cells containing dermal papilla and dermal sheath from patients; culturing said cells as obtained from patients using condition medium collected from the prior step; injecting cultured cells in the predetermined amount and the predetermined ratio of dermal papilla cells and dermal sheath cells in the predetermined amount area characterized in that the step of injecting and collecting condition medium from said keratinocytes comprises the step of applying a solution obtained from the mixture of defined keratinocyte-serum free medium (DK-SFM) and Mesenchymal stem cell culture medium (MSC) to said initial keratinocytes, removing defined keratinocyte-serum free medium (DK-SFM), and extracting a condition medium from the said mixture and wherein the step of culturing hair follicle cells as obtained from patients comprises the step of applying condition medium extracted and MSC in the ratio of 1:1.

[0016] In one aspect, defined keratinocyte-serum free medium (DK-SFM) comprises DK SFM basal medium + 0.2% DK-SFM growth supplement + 100 U / ml penicillin and 100 pg / ml streptomycin.

[0017] In the other aspect, Mesenchymal stem cell culture medium (MSC) comprises alpha MEM, 10% of Human serum, 2mM of L-glutamine, 100 U / ml of penicillin and 100 pg / ml of streptomycin and the ratio of defined keratinocyte-serum free medium (DK-SFM) and Mesenchymal stem cell culture medium (MSC) is in the range of 1:1 to 3:1.

[0018] In the other aspect, initial keratinocytes are cells obtained from amnion. 12 cell lines are derived from dermal papilla and the number of cells per vial is 2 X 106 cells per vial.

[0019] In the other aspect, the step of collecting hair follicle cells containing dermal papilla and dermal sheath from patients comprises the step of collecting hair follicle cells in the amount of 20 to 80 follicle per patient; the step of placing collected hair follicle cells on gauze impregnated with Hypothermosol; and the step of transporting said preserved cells.

[0020] In the other aspect, the step of transporting said preserved cells comprises the step of controlling said preserved cells at the temperature in the range of 4°C to 25°C and the transportation is completed within the duration of 24 hours.

[0021] In the other aspect, the step of culturing said cells as obtained from patients using condition medium collected from the prior step comprises the step of covering dermal papilla and dermal sheath with the provided slide. The provided slide may have a width of 2.5 centimeters and the length in the range of 1 to 2.5 centimeters and the thickness in the range of 1.0-1.2 millimeters and there can be 7 to 10 dermal papilla or dermal sheath per slide.

[0022] In the other aspect, the step of injecting cultured cells in the predetermined amount and the predetermined ratio of dermal papilla cells and dermal sheath cells in the predetermined amount area is the step of injecting cultured cells in the amount of 100,000 to 200,000 having the ratio between dermal papilla and dermal sheath of 1:1 in the area of 1 square centimeter with the predefined solution in the amount of 0.5 milliliters.

[0023] The objectives and unique characteristics and other aspects of this invention will be described in more detail by way of the examples and drawings included and the best mode will also be further described.

[0024] Brief Descriptions of the Drawings

[0025] Figure 1 shows Hair Follicle Extraction Process according to this invention.

[0026] Figure 2 shows Dermal Papilla and Dermal Sheath cells extraction method.

[0027] Figure 3 shows the numerous new formed hairs

[0028] Detailed Descriptions of the Preferred Embodiments

[0029] The descriptions in more detail of this invention will be provided by way of examples in order to make the descriptions clearer. Parts that are the same in these drawings will be identified by the same referenced number. The said descriptions in more detail, however, are provided without any limitation to the scope of the invention.

[0030] In this regard, this invention can be implemented or applied in various manners. This disclosure will present the different examples as references for implementations or applications having the same characteristics. Nevertheless, as mentioned above, these are provided without any limitation to the scope of the invention.

[0031] The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle comprises the steps of: providing initial keratinocytes to be used for creating condition medium; creating and collecting condition medium from said keratinocytes; collecting hair follicle cells containing dermal papilla and dermal sheath from patients; culturing said cells as obtained from patients using condition medium collected from the prior step; injecting cultured cells in the predetermined amount and the predetermined ratio of dermal papilla cells and dermal sheath cells in the predetermined amount area characterized in that the step of injecting and collecting condition medium from said keratinocytes comprises the step of applying a solution obtained from the mixture of defined keratinocyte-serum free medium (DK-SFM) and Mesenchymal stem cell culture medium (MSC) to said initial keratinocytes, removing defined keratinocyte-serum free medium (DK-SFM), and extracting a condition medium from the said mixture and wherein the step of culturing hair follicle cells as obtained from patients comprises the step of applying condition medium extracted and MSC in the ratio of 1:1.

[0032] It is preferable to have defined keratinocyte-serum free medium (DK-SFM) which comprises DK SFM basal medium, 0.2% DK-SFM growth supplement, 100 U / ml penicillin and 100 pg / ml streptomycin. Mesenchymal stem cell culture medium (MSC) may also comprise alpha MEM, 10% of Human serum, 2mM of L-glutamine, 100 U / ml of penicillin and 100 pg / ml of streptomycin and the ratio of defined keratinocyte-serum free medium (DK-SFM) and Mesenchymal stem cell culture medium (MSC) is in the range of 1:1 to 3:1.

[0033] For the step relating to collecting hair follicle cells containing dermal papilla and dermal sheath from patients, initial keratinocytes can be cells obtained from amnion. 12 cell lines can be derived from dermal papilla and the number of cells per vial is 2 X 106 cells per vial.

[0034] For the step of collecting hair follicle cells containing dermal papilla and dermal sheath from patients, this step may refer to the procedure of collecting hair follicle cells in the amount of 20 to 80 follicle per patient; the step of placing collected hair follicle cells on gauze impregnated with Hypothermosol; and the step of transporting said preserved cells.

[0035] For the step of transporting said preserved cells, this may comprise the step of controlling said preserved cells at the temperature in the range of 4°C to 25°C and the transportation is completed within the duration of 24 hours.

[0036] Additionally, the step of culturing said cells as obtained from patients using condition medium collected from the prior step may comprise the step of covering dermal papilla and dermal sheath with the provided slide. The provided slide may have a width of 2.5 centimeters and the length in the range of 1 to 2.5 centimeters and the thickness in the range of 1.0-1.2 millimeters and there can be 7 to 10 dermal papilla or dermal sheath per slide. For the step of injecting cultured cells in the predetermined amount and the predetermined ratio of dermal papilla cells and dermal sheath cells in the predetermined amount area, it is preferable to inject cultured cells in the amount of 100,000 to 200,000 having the ratio between dermal papilla and dermal sheath of 1:1 in the area of 1 square centimeter with the predefined solution in the amount of 0.5 milliliters. Based on this disclosure, the number of hair follicle cells can increase to 50 million cells based on the collection of 50 hairs from a patient.

[0037] The table below highlights benefits of the method according to this disclosure which is named as "Follicular hair restoration" or "FHR" in shot.

[0038] As per the table, there are various benefits over the traditional methods. These include the long-term storage for preserving healthy hair, natural growth of hair stalk, emergence of new healthy follicle. The method as disclosed is also accessible by most people and there is no need for skillful transplant doctors. The method can be reproduced and implemented in a repetitive manner. The economic benefits including the shorter operative time, and lower costs for the required physicians or technical staff can also be realized. Details of which will be elaborated below.

[0039] According to the existing transplant methods including FUE and FUT, such methods need to use graft immediately. This is different from the method according to this disclosure which covers the preservation by way of freezing using liquid nitrogen which leads to the ability to grow well upon thawing.

[0040] In this regard, hair follicles, including both dermal papilla and dermal sheath, should not be cultured for more than three passages. After three passages of expansion, cells will lose hair growth properties. Cells are divided into multiple vials so that physicians can plan with patient for its usage.

[0041] According to this disclosure, due to the small amount of hair needed during harvesting which is around 20 to 80 follicles, multiple hair harvest can be conducted numerously. This surpasses the limitation of the previous or existing methods in which only two times of hair harvesting can be conducted. Other advantages which overcome the prior limitations based on the previous or existing method include the implantation time and implementation area. According to the present disclosure, patient can maintain fully hair grow for throughout life and has no limitation with regard to implantation area. and exiting methods may still be noticeable due to unnatural straight line on the forehead. According to our present disclosure, such step is not required as hairs will emerge naturally from seeding leading to natural look. Furthermore, with the method according to this disclosure, roots are deeper and more difficult to drop off when compared to the strength obtained via the previous and existing methods.

[0042] During the actual operations, operations based on the previous and existing methods can cause back and neck pain for the physicians. This is due to the long working hours of hair plucking and retransplant. Physicians also need to have experience and expertise in order to provide a good-looking hair transplant. With the method according to this disclosure, this method requires only the injection back onto the scalp within 30 minutes. This makes the procedure readily to everyone and can be conducted everywhere without the need for skillful experienced physicians.

[0043] Although this invention is described in detail, it will be understood that this invention is intended to cover any modification or solutions provided by the persons skilled in the arts. The scope of this invention is based on the claims and also cover any characteristics of the invention that may not be specifically covered in the claims, but still cause the same effects as those of this invention.

[0044] Best Mode of the Invention

[0045] As disclosed in the Disclosure of the Invention.

Claims

Claims1. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle comprising the steps of: providing initial keratinocytes to be used for creating condition medium; creating and collecting condition medium from said keratinocytes; collecting hair follicle cells containing dermal papilla and dermal sheath from patients; culturing said dermal papilla and dermal sheath cells as obtained from patients using condition medium collected from the prior step; injecting cultured cells in the predetermined amount and the predetermined ratio of dermal papilla cells and dermal sheath cells in the predetermined amount area characterized in that the step of injecting and collecting condition medium from said keratinocytes comprises the step of applying a solution obtained from the mixture of defined keratinocyte-serum free medium (DK-SFM) and the first Mesenchymal stem cell culture medium (MSC) to said initial keratinocytes, removing defined keratinocyte-serum free medium (DK-SFM), and extracting a condition medium from the said mixture and wherein the step of culturing dermal papilla and dermal sheath cells as obtained from patients comprises the step of applying condition medium extracted and the second MSC in the ratio of 1:

12. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 1 wherein defined keratinocyte-serum free medium (DK-SFM) comprises DK SFM basal medium + 0.2% DK-SFM growth supplement + 100 U / ml penicillin and 100 pg / ml streptomycin.

3. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 2 wherein Mesenchymal stem cell culture medium (MSC) comprises alpha MEM, 10% of Human serum, 2mM of L-glutamine, 100 U / ml of penicillin and 100 pg / ml of streptomycin and the ratio of defined keratinocyte-serum free medium (DK-SFM) and the first Mesenchymal stem cell culture medium (MSC) is in the range of 1:1 to 3:1.

4. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 1 wherein the step of culturing dermal papilla and dermal sheath hair follicle cells as obtained from patients comprises the step of applying condition medium extracted and the second MSC in the ratio of 1:1 and alpha MEM, 10% of Human serum, 2mM of L-glutamine, TGF-beta 1, TGF-beta 2, IGF-I, 100 U / ml of penicillin.

5. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 1, 2 or 3 wherein initial keratinocytes are cells obtained from amnion.

6. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 1 wherein 12 cell lines are derived from dermal papilla and the number of cells per vial is 2X106 cells per vial.

7. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 1 or 5 wherein the step of collecting hair follicle cells containing dermal papilla and dermal sheath from patients comprises the step of collecting hair follicle cells in the amount of 20 to 80 follicle per patient; the step of placing collected hair follicle cells on gauze impregnated with Hypothermosol; and the step of transporting said preserved cells.

8. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 7 wherein the step of transporting said preserved cells comprises the step of controlling said preserved cells at the temperature in the range of 4°C to 25°C and the transportation is completed within the duration of 24 hours.

9. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 8 wherein the step of controlling said preserved cells at the temperature in the range of 4°C to25°C comprises the step of providing a liquid nitrogen tank and the step of controlling the number of cells per vial for storage in said tank is 2. Ox 106 cells per vial.

10. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 1 wherein the step of culturing dermal papilla and dermal sheath cells as obtained from patients using condition medium collected from the prior step comprises the step of covering dermal papilla and dermal sheath with the provided slide.

11. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 8 wherein the provided slide has a width of 2.5 centimeters and the length in the range of 1 to 2.5 centimeters and the thickness in the range of 1.0-1.2 millimeters and there are 7 to 10 dermal papilla or dermal sheath per slide.

12. The method of hair transplant using dermal papilla and dermal sheath obtained from hair follicle of claim 1 wherein the step of injecting cultured cells in the predetermined amount and the predetermined ratio of dermal papilla cells and dermal sheath cells in the predetermined amount area is the step of injecting cultured cells in the amount of 100,000 to 200,000 having the ratio between dermal papilla and dermal sheath of 1:1 in the area of 1 square centimeter with the predefined solution in the amount of 0.5 milliliters beneath the dermis layer.

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