Microneedle based cell delivery

Abstract

The invention relates to a device for use in skin improvement or repair, including promoting hair growth, comprising the use of microneedles for the transplantation of cells and a method employing the use of same.

Claims

1. A device for skin improvement or repair comprising: a plurality of microneedles attached to or integral with a supporting base member and arranged in at least one circular pattern on same wherein said microneedles are hollow and have a bore size of between 75-150 m diameter and a length of between 250 m and 1000 m, wherein the device is adapted for (a) extracting cells from a first area of skin of an individual, and subsequently (b) injecting said cells into a second area of skin of said individual, wherein there is no observed loss of viability of said cells as a result of the transplantation process; and whereby said second area of skin is improved by the transplantation of said cells.

2. The device according to claim 1 wherein in step (b), said cells are injected into the viable epidermis, papillary.

3. The device according to claim 1 wherein said microneedles have a bore size of 75-150 m diameter.

4. The device according to claim 1 wherein a plurality of concentric circular patterns of microneedles is provided on said base member.

5. The device according to claim 4 wherein two concentric circular patterns of microneedles are provided on said base member.

6. The device according to claim 1 wherein said microneedles are attached to or integral with said base member so that their longitudinal axis is normal to the supporting axis of said base member or so that their longitudinal axis is at an angle to the supporting axis of said base member such that said microneedles splay outwards with respect to the supporting axis of said base member.

7. The device according claim 1 wherein between 6 and 48 microneedles are used in each of said circular patterns.

8. The device according to claim 1 wherein 24 microneedles are used in an outer concentric circular pattern and 12 microneedles are used in an inner concentric circular pattern.

9. The device according to claim 1 wherein said microneedles are between 250 m and 1000 m in length.

10. The device according to claim 1 wherein said microneedles are about 750 m in length.

11. The device according to claim 1 wherein said microneedles are made from a polymer, co-polymer, polysaccharide, sugar, silicon or steel.

12. A method for skin improvement or repair comprising: a) extracting with a plurality of microneedles attached to a supporting base member and arranged in at least one circular pattern on same at least one cell from a first area of skin of an individual to be treated; and b) injecting with at least one hollow microneedle, having a bore size of 75-150 m and a length of between 250 m and 1000 m, said cell(s) into a second area of skin; whereby said second area of skin is improved by the transplantation of said cell(s) therein wherein there is no observed loss of viability of said cells as a result of the transplantation process; and whereby said second area of skin is improved by the transplantation of said cells.

13. The method according to claim 12 wherein said bore size is between 75 and 150 m diameter.

14. The method according to claim 12 wherein said injecting also involves the use of a single microneedle or a plurality of microneedles attached to a supporting base member and where a plurality of microneedles are used they are arranged in at least one row, rectangular array or circular pattern on same.

15. The method according to claim 12 wherein said cell(s) are selected from the group comprising: melanocytes, keratinocytes, dermal fibroblasts, corneocytes, Langerhans cells, dermal dendritic cells, epidermal stem cells, Merkel cells, mast cells, macrophages, T-cells, dermal sheath cells or follicular outer root sheath cells.

16. The method according to claim 12 wherein in step b) said cell(s) are injected into the viable epidermis, papillary dermis or reticular dermis layers of the skin.

17. The method according to claim 12 wherein the microneedles used in step a) are different to that/those used in step b).

18. The method according to claim 12 wherein said method is repeated for all areas of the skin for which repair or improvement is desired.

19. The method according to claim 12 wherein said cell(s) extracted in step a) are preserved prior to the performance of step b).

20. The method according to claim 19 wherein said cell(s) extracted in step a) are preserved prior to the performance of step b) and further, wherein said preserved cell(s) are used for repeated procedures whereby measured amounts of the preserved cells are repeatedly used for the repeated performance of step b).

21. The method of claim 12 wherein no loss of cell viability occurs.

Description

(1) The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein:

(2) FIG. 1. Methylene blue staining to evaluate the diffusion of liquid through skin following microneedle injection;

(3) FIG. 2. [A] Keratinocytes in culture, with the typical cobblestone appearance, [B] Melanocytes in culture, with the typical dendritic phenotype;

(4) FIG. 3. Number of total keratinocytes counted after passing through microneedles with different bore sizes, divided into alive (blue) and dead (red) cells. The error bars represent the standard error on the means obtained on 3 repeats;

(5) FIG. 4. Number of total melanocytes counted after passing through microneedles with different bore sizes, divided into alive (blue) and dead (red) cells. The error bars represent the standard error on the means obtained on 3 repeats;

(6) FIG. 5. Keratinocytes in culture at 48 h after passing through microneedles with different bore sizes: 75 m [A], 100 m [B] and 150 m [C];

(7) FIG. 6. Melanocytes in culture at 48 h after passing through microneedles with different bore sizes: 75 m [A], 100 m [B] and 150 m [C];

(8) FIG. 7. Hooked microneedles for cell extraction before (top row) and after (bottom row) skin penetration, viewed using Scanning Electron Microscopy;

(9) FIG. 8. Use of hollow microneedles for cell extraction, viewed using Scanning Electron Microscopy;

(10) FIG. 9. Skin cells in culture at 96 h after collection from human skin by microneedle scraping;

(11) FIG. 10. Human keratinocyte cells (stained blue with Hoechst staining) injected using microneedles at a concentration of 500,000 cells/50 l into excised human breast skin. Skin was imaged using light and fluorescence microscopy. Skin autofluorescence appears as green. The white line denotes the skin surface;

(12) FIG. 11. Shows pictures of the proprietary designs used to work the invention;

(13) FIG. 12. Arrangement of microneedles on a supporting base member Design E and F; Disk diameter=10 mm; Number of needles per disk=24; Needles length=750 m; Material: Stainless Steel; In design E the needles are in plane; In design F the needles are bent out of plane; Needles manufactured by wire Electrical Discharge Machining (EDM);

(14) FIG. 13. A further arrangement of microneedles on a supporting base member, Design G: Outer disk diameter=10 mm; Number of needles in the outer disk=24; Inner disk diameter=5 mm; Number of needles in the inner disk=12; Total number of needles=36; Needles length=750 m; Material: Stainless steel; The needles are all bent out of plane; Manufactured by wire EDM; The arrays are stacked and so the number of needles could be expanded easily;

(15) FIG. 14. Shows images of the survival of extracted cells after passing through microneedles was also assessed. All the cells survived after passing through hollow microneedles with a bore size75 m;

(16) FIG. 15. Upper image shows that Hoechst labelled cells (blue nuclei) were injected in skin explants and successfully delivered to the dermis; lower image shows that the injected cells (blue nuclei) maintained their phenotype after injection in skin. Cells with a green cytoplasm are melanocytes, while cells with a red cytoplasm are keratinocytes; and

(17) Table 1 contains information relating to the efficiency of cell extraction using the specified microneedles, together with the technical features of each microneedle design.

(18) Materials and Methods

(19) Cell Source

(20) Melanocytes and keratinocytes were isolated from non-affected skin (biopsy or cell scraping) or from the hair follicles. These were used directly or cultured to increase cell numbers.

(21) Additionally, microneedle injection of primary melanocytes and keratinocytes obtained from commercial sources (Life Technologies) was also undertaken.

(22) Cell Culture

(23) Melanocytes and keratinocytes were expanded in vitro, using selective growth media.

(24) For melanocytes, the selective growth media was Medium 254 (Life Technologies) or equivalent, supplemented with Human Melanocyte Growth Supplement-2, PMA-free (Life Technologies) or equivalent.

(25) For keratinocytes, the selective growth media was Epi-Life Medium (Life Technologies) or equivalent, supplemented with Human Keratinocyte Growth Supplement (Life Technologies) or equivalent.

(26) Cells were incubated at 37 C. in 5% CO.sub.2 and media replaced every 48 to 72 h until a sufficient number of cells were obtained (determined depending on the extension of the area to be treated, approximately 10.sup.5 melanocytes/cm.sup.2).

(27) Extraction of viable live cells from the skin was assessed using light microscopy.

(28) Cells were delivered to freshly excised human breast skin. A fraction of the cell culture was also stored in liquid nitrogen for subsequent application if needed, removing the need to repeat the isolation step.

(29) Microneedle Delivery

(30) Cells were delivered to the recipient site by the use of specifically designed microneedles. According to preliminary studies, using a marker dye instead of a cell suspension (FIG. 1), approximately 100 L of a 10.sup.6 cells/mL solution was sufficient to re-pigment a skin area of approximately 1 cm.sup.2, delivering 10.sup.5 cells to the area.

(31) Microneedle Specifications

(32) The microneedles used were hollow silicon microneedles, arranged in an array, to cover an area of approximately 0.5 cm.sup.2.

(33) Microneedles had a bore size between 75 and 150 m, a wall thickness between 50 and 150 m, spacing between 500 and 1000 m, and a length between 300 and 700 m.

(34) Cell Extraction Using Microneedles

(35) Different types of microneedles were used to perform cell extraction from skin. These included commercially available silicon microneedles (A, B, C) and our proprietary stainless steel microneedles (D, E, F, G).

(36) Pictures of the proprietary stainless steel microneedle designs (D, E, F, G) are shown in FIG. 11. The specifications of these needles are as follow.

(37) Design D: Array width=1.1 cm; Number of needles per array=10; Needles length=between 350 and 420 m; Number of arrays stacked=3; Total number of microneedles=30; Material: Stainless Steel. See FIG. 11D (made by Electrical Discharge Machining (EDM))

(38) Design E and F: Disk diameter=10 mm; Number of needles per disk=24; Needles length=750 m; Material: Stainless Steel; In design E the needles are in plane; In design F the needles are bent out of plane; Needles manufactured by wire Electrical Discharge Machining (EDM). See FIGS. 11E and F, and FIG. 12.

(39) Design G: Outer disk diameter=10 mm; Number of needles in the outer disk=24; Inner disk diameter=5 mm; Number of needles in the inner disk=12; Total number of needles=36; Needles length=750 m; Material: Stainless steel; The needles are all bent out of plane; Manufactured by wire EDM; The arrays are stacked and so the number of needles could be expanded easily. See FIG. 11G and FIG. 13.

(40) How the Microneedles are Used on Skin

(41) Design A, B, C, and D: A 2 cm.sup.2 area of the skin surface is scraped multiple time with a linear movement (left to right and right to left).

(42) Design E: The disk is rolled multiple times on a 2 cm.sup.2 area of the skin surface.

(43) Design F and G: A 2 cm.sup.2 area of the skin surface is scraped multiple times with a circular movement (clockwise).

(44) After scraping, the material collected on the microneedles is transferred to a tube containing cold trypsin and incubated overnight at 4 C. The following day, the trypsin is inactivated with the same volume of serum. The cell containing solution is then filtered and centrifuged, and cells are resuspended, ready for injection.

(45) With this method, different types of cells are extracted, including keratinocytes, melanocytes, fibroblasts, Merkel cells, Langerhans cells, macrophages, adipocytes, dendritic cells, etc.

(46) The efficiency of cell extraction using these microneedles was assessed and it is reported in table 1 below, together with the technical features of each microneedle design.

(47) After extraction, culturing the cells in a specific growth media (Medium 254 from Life Technologies or equivalent, supplemented with Human Melanocyte Growth Supplement-2, PMA-free from Life Technologies or equivalent) promotes melanocytes to differentiate from the pool of extracted cells, as shown in FIG. 14.

(48) The survival of extracted cells after passing through microneedles was also assessed. All the cells survived after passing through hollow microneedles with a bore size75 m.

(49) Data Regarding Cell Injection

(50) Cell survival has been tested after passing suspensions ranging from 10.sup.5 to 10.sup.7 cells/mL through different types of hollow microneedles. Cell survival is near 100% when cells at all these concentrations are injected thorough hollow microneedles with a bore size75 m. Cell survival is significantly reduced if the cells are passed through hollow microneedles with smaller bore size, dropping to approximately 50% when the bore size is 50 m.

(51) Cell adhesion, cell proliferation, and cell phenotype are maintained after passing through microneedles with a bore size75 m at all the concentrations tested.

(52) Cell delivery to skin can be performed efficiently, with cells maintaining their original phenotype once injected, as shown in FIG. 15.

(53) Results

(54) Cell Delivery Through Microneedles

(55) Cell cultures from commercially available human epidermal keratinocytes and melanocytes were established, and culturing conditions were optimized (FIG. 2).

(56) Cell survival after passing through microneedles with different bore diameters (50, 60, 75, 100 and 150 m) was tested using a 10.sup.5, 10.sup.6, 210.sup.6 and 10.sup.7 cells/mL suspension. Trypan Blue staining was used to confirm cell viability. Both keratinocytes (FIG. 3) and melanocytes (FIG. 4) survived the procedure with all the concentrations tested when the bore diameter was greater than 60 m. There was also no loss of cells during the procedure, showing that cells are not retained inside the microneedles.

(57) The ability of cells to adhere to a surface and proliferate after passing through microneedles with different bore sizes (75, 100 and 150 m) was tested. Both keratinocytes (FIG. 5) and melanocytes (FIG. 6) were able to attach to culture plates and proliferate after the procedure.

(58) Cell Extraction from Skin Using Microneedles

(59) We also used prototype microneedles to test the ability of microneedles to extract cells from the skin.

(60) Firstly, we tested the ability of hooked stainless steel microneedles to extract skin cells after skin penetration (excised human breast skin) and removal (FIG. 7).

(61) We also tested hollow microneedles using the same approach (FIG. 8).

(62) Subsequently, as proof-of-concept, we also tested the ability of different types of solid and hollow microneedles to collect skin cells by scraping along the skin's surface (excised human breast skin). After scraping the skin, cells were detached from the microneedles by rinsing in culture media and captured in a culture plate.

(63) Microneedle extraction of cells surprisingly allowed us to capture and culture a significant number of skin cells (FIG. 9).

(64) In further studies we tested whether microneedles can deliver cells into relevant skin compartments. In these studies human keratinocytes were labelled prior to skin injection (excised human breast skin). Hollow microneedle delivery of 5000, 50000 and 500000 cells into excised human skin resulted in deposition of the cells in the upper dermis (FIG. 10).

(65) Different types of microneedles were used to perform cell extraction from skin. These included commercially available silicon microneedles (A, B, C) and our own proprietary stainless steel microneedles (D, E, F, G).

(66) Pictures of the stainless steel proprietary designs are shown in FIG. 11 with schematics showing the microneedles designs used in E, F and G shown in FIGS. 12 and 13.

(67) After extraction, culturing the captured cells in a specific growth media promotes melanocytes to differentiate from the pool of extracted cells, as shown in FIG. 14 for designs C, D, E, F and G. The survival of extracted cells after passing through microneedles was also assessed. All the cells survived after passing through hollow microneedles with a bore size75 m.

(68) Cell adhesion, cell proliferation, and cell phenotype are maintained after passing through microneedles with a bore size75 m at all the concentrations tested.

(69) As further evidence of efficient cell delivery using microneedles, cells (with a blue nuclear cell staining) maintain their original phenotype once injected, as shown in FIG. 15.

SUMMARY

(70) In summary our studies have shown that:

(71) 1) Cells can be collected from skin using microneedles;

(72) 2) Cells can be injected through hollow microneedles with a variety of bore diameters. All of the cells are injected with none retained, with high viability (cell survival is near 100%);

(73) 3) Cells can be injected into skin using microneedles.

(74) This disclosed method therefore paves the way for a new minimally-invasive and pain-free approach wherein cells can be extracted and delivered to the various layers of the skin using microneedles, with little or no recovery time required.

(75) TABLE-US-00001 Number Are of cells melano- Type of extracted cytes microneedles De- by extracted used for Length of sign scraping a and extraction microneedles name Material 2 cm.sup.2 area viable? Hollow, 50 m 450 m A Silicon 5 10.sup.5 NO bore size Hollow, 60 m 600 m B Silicon 4 10.sup.5 NO bore size Hollow, 80 m 750 m C Silicon 6 10.sup.5 YES bore size 2 cells (0.0003%) Solid, 3 rows 350 to 420 m D Steel 5 10.sup.5 Yes of 10 12 cells microneedles (0.0024%) Solid, disk of 750 m E Steel 6 10.sup.5 YES 24 needles - 2 cells in plane (0.0003%) Solid, disk of 750 m F Steel 8 10.sup.5 YES 24 needles - approxi- out of plane mately 120 cells (0.015%) Solid, 2 750 m G Steel 1 10.sup.6 YES concentric approxi- disks, total of mately 36 needles - 300 cells out of plane (0.03%)