KR102522974B1 - 2d cell and 3d cell implant patch - Google Patents

Abstract

It relates to a patch carrying two- and three-dimensionally co-cultured cells, which is manufactured by two-dimensionally and three-dimensionally co-cultivating cells on a sheet having an array structure formed by arranging a plurality of well units of the present invention. contains two-dimensional cell sheets and three-dimensional cell spheroids together, increases the secretion of angiogenic factors and growth factors related to wound healing, and exhibits high survival and engraftment rates, and separate collection steps after culturing cells Since it can be delivered directly without a patch, the cell delivery rate is high and the manufacturing efficiency is increased, so it can be usefully used as a patch for tissue regeneration.

Description

Patch for 2D cell and 3D cell transplant {2D CELL AND 3D CELL IMPLANT PATCH}

The present invention relates to a patch carrying two-dimensional and three-dimensional co-cultured cells.

Various organs and tissues of the human body are composed of various types of cells, and maintain homeostasis through the interaction of each cell. The skin is a tissue that covers the outermost part of the human body and serves as a primary defense function to protect the body from external chemical/physical shocks by protecting the human body and functioning as a barrier. The skin is composed of three layers: epidermis, dermis, and subcutaneous fat layer. In the case of skin, it is typically composed of various cells such as fibroblasts and keratinocytes, and when the skin is damaged, it recovers through the stage of wound healing. Wound healing is a tissue repair process in response to damaged skin. It is a complex biological process including chemotaxis, cell differentiation and replication, lipoprotein synthesis, and angiogenesis. It is done. Wound healing phases include (1) vascular response (hemostasis), (2) inflammatory response (inflammation), proliferative phase (also known as granulation, epithelialization, and active growth phase) and (4) maturation (remodeling). remodeling) step, where these steps overlap to reform the extracellular matrix (H. J. Lim, H. T. Kim, E. J. Oh, J. H. Chio, H. D. Ghim, D. G. Pyun, S. B. Lee, D. J. Chung, and H. Y. Chung). , Polymer (korea), 34, 363, 2010. Immune action initiated by macrophages as well as fibroblasts and keratinocytes at each stage (e.g. macrophage M2 polarization), and endothelial cells, epithelial cells ( It recovers the wound site through angiogenesis through the interaction of various cells such as epithelial cells and platelet cells.

Conventionally, cell injection therapy and transplantation after 3D cell culture have been used for wound healing, but in the case of cell injection therapy, the affected area where cells are transplanted has an environment in which cells are engrafted and it is difficult to show a therapeutic effect, so low survival rate and engraftment rate , and cannot directly deliver cells to severely damaged tissue, so it does not reach the level of clinical efficacy verification due to the limitation of not delivering cells with therapeutic effects to the center of the affected area. In addition, in the case of the conventional transplantation method after 3D cell culture, there is a time and labor intensive problem because it requires additional processes including a collection step for injection after spheroids are formed, and in the case of non-adherent cells, it is difficult to culture spheroids. In the cell collection stage, there are many cases where the shape collapses due to shear stress, etc., so it has been difficult to deliver it to the affected area in an intact state.

On the other hand, in the case of cells, viability, growth factors, and various cytokine secretion patterns change according to culture in 2D and 3D. It is known that adult stem cells, fibroblasts, and keratinocytes secrete more angiogenesis-related factors when cultured in 3D than in 2D. It is also known that the ability to secrete pro- or anti-inflammatory cytokines that directly affect the primary immune inflammatory response depends on the cell structure. Cases of grafting 2D or 3D cells to tissue regeneration have recently been actively studied. However, studies are being conducted in which most of one form is combined with various supports, inorganic substances, genes, growth factors, etc., and 2D and 3D forms of cells are delivered directly to the affected area at the same time without inflow of external substances to regulate blood vessel regeneration, inflammation, and immune action No cases of attempted tissue regeneration have been reported yet.

An object of the present invention is to provide a method for co-cultivating cells in two dimensions and three dimensions.

Another object of the present invention is to provide a patch for wound healing.

In addition, an object of the present invention is to provide a method for manufacturing a patch for wound healing.

In order to achieve the above object, the present invention provides a method for two-dimensionally and three-dimensionally co-cultivating cells on a sheet having an array structure formed by arranging a plurality of well units.

In addition, the present invention provides a patch for wound healing.

In addition, the present invention provides a method for manufacturing a patch for wound healing.

The patch fabricated by two- and three-dimensional co-culture of cells on a sheet having an array structure formed by arranging a plurality of well units of the present invention includes a two-dimensional cell sheet and a three-dimensional cell spheroid. The secretion of angiogenic factors and growth factors related to wound healing increases, shows a high survival rate and engraftment rate, and can be delivered immediately without a separate collection step after culturing cells, which has the effect of increasing cell delivery rate and manufacturing efficiency. It can be usefully used as a patch for playback.

1 is a diagram showing a structure of a PDMS-based patch CSCP and a process for 2D and 3D co-culture of human dermal fibroblasts (hDF) on a PDMS patch, and a diagram showing cell images after culture.
Figure 2 is a diagram confirming the expression of genes related to angiogenesis, pro-inflammatory cytokines and anti-inflammatory cytokines in Cell, SP and FP groups.
Figure 3 shows the culture medium of 2D and 3D fibroblast co-culture CSCP (FP) (blue bar), 2D fibroblast only group (Cell) (red bar) and 3D fibroblast only group (SP) (purple bar). It is a diagram confirming gene expression of m1 and m2 macrophage markers in m1 macrophages.
Figure 4 is a diagram confirming the wound healing effect due to transplantation of CSCP loaded with 2D and 3D co-cultured fibroblasts in a wound-induced mouse model:
NT: group of mice treated with nothing;
FP: CSCP-transplanted mice group loaded with 2D and 3D co-cultured fibroblasts;
Cell: mouse group directly injected with 2D cultured fibroblasts alone;
SP: group of mice directly injected with 3D cultured fibroblasts alone; and
PDF: Groups of mice directly injected with 2D cells and 3D cells removed from CSCP patches.
Figure 5 is a diagram showing the difference in culture of TCP and CSCP of keratinocytes that are difficult to culture hanging drops due to lack of adhesive properties.
6 is a diagram confirming the cell viability of keratinocytes cultured under hypoxic conditions by performing live and dead staining using a fluorescence microscope.
7 is a diagram confirming the expression levels of cell proliferation-related factors, apoptosis-related factors, and angiogenesis factors in cells of CSCP and TCP.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

In one aspect, the present invention provides a method for two-dimensionally and three-dimensionally co-cultivating cells on a sheet having an array structure formed by arranging a plurality of well units: cells are grown in two dimensions on the side of the sheet having well openings. incubate; and a method for culturing cells in three dimensions inside a well.

In one embodiment, the sheet may be made of a biocompatible material, and the biocompatible material may be at least one selected from the group consisting of polydimethylsiloxane (PDMS), silicone, polyethyleneglycol, and alginate. can

In one embodiment, the cells are tissue-derived from any cell, such as cells from heart tissue, skeletal muscle tissue, smooth muscle tissue, liver tissue, kidney tissue, cartilage tissue, skin, bone marrow tissue, or combinations of these tissues. can be used together The cells may be sourced anywhere, including from any commercial source, or even from an individual subject or patient. For example, a cell of the invention can be a commercially available cell line, a cell obtained directly from a subject, eg, a cell isolated from a biopsy. In other embodiments, the cells can be a mixture of cells that are different from one another, such a mixture of cells being a mixture of healthy or diseased cells from the same or different tissues, a mixture of cells from different sources or a patient, or both from a patient. and mixtures of cells from commercial sources. The cell may also be a genetically engineered cell, such as a drug-resistant or drug-sensitive engineered cell line, or other types of genetically engineered cells, including those expressing various biomarkers such as GFP.

In one embodiment, the cells are most preferably human or mammalian cells, and may be fibroblasts, keratinocytes, cardiac fibroblasts, cardiomyocytes, stem cells or primary cultured cells. The primary cultured cells may be human skin-derived keratinocytes or human skin-derived fibroblasts. The stem cell or primary cultured cell is not particularly limited to its origin as long as it is a cell that can be cultured by the cell culture method and cell cultivator of the present invention, and examples thereof include human, monkey, pig, horse, cow, sheep, dog, and cat. , mouse, and rabbit-derived cells. The stem cells or primary cultured cells are preferably human-derived stem cells or primary cultured cells, but are not limited thereto.

In one embodiment, the density of the cells may be 10 ⁵ to 10 ⁶ cells/ml, preferably 3x10 ⁵ to 8x10 ⁵ cells/ml, and more preferably 5x10 ⁵ to 6x10 ⁵ cells/ml.

In one embodiment, the 2D cultured cells and the 3D cultured cells may be the same cell, different cells, or a combination of different cells.

In one embodiment, the cells may be adherent or non-adherent cells.

In one embodiment, the wells of the sheet and the side having the openings of the wells may be immersed in a cell culture medium.

In one embodiment, when cells are cultured in two dimensions, cell sheets can be formed, and when cells are cultured in three dimensions, cell spheroids can be formed.

In one embodiment, it may comprise 2x10 ³ to 3x10 ⁴ cells per cell spheroid.

As used herein, the term "stem cell" refers to an undifferentiated cell having self-replicating and differentiated proliferation. Stem cells include subgroups such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation ability. The pluripotent stem cells refer to cells having the ability to differentiate into all tissues or cells constituting the living body, and the pluripotent stem cells have the ability to differentiate into multiple types of tissues or cells, although not all types. means cells with Unipotent stem cells refer to cells having the ability to differentiate into specific tissues or cells. Examples of pluripotent stem cells include embryonic stem cells (ES Cell), undifferentiated germline cells (EG Cell), iPS cells, and the like. Bone marrow-derived, umbilical cord blood or umbilical cord-derived, etc.), hematopoietic stem cells (derived from bone marrow or peripheral blood, etc.), nervous system stem cells, and adult stem cells such as reproductive stem cells. Hepatocytes (Committed stem cells), which exist in a low state and produce only hepatocytes through vigorous division after activation, may be mentioned.

In the present invention, the term "primary cell" is a cell isolated from a tissue extracted from an individual without manipulation of genes, etc., and represents a function of a living organ/tissue. Primary cultured cells are isolated from skin, vascular endothelium, bone marrow, fat, cartilage, etc., and are used as cell therapy products to study the functions of tissues and cells or to restore lost tissues.

As used herein, the term "PDMS" refers to the polymer poly(dimethylsiloxane). Polydimethylsiloxane (PDMS) belongs to a group of polymeric organosilicon compounds commonly referred to as silicones. PDMS is the most widely used silicone-based organic polymer and is particularly known for its unique rheological (or flow) properties. PDMS is optically transparent and is generally inert, non-toxic, and non-flammable. Also called dimethicone, it is one of several types of silicone oils (polymerized siloxanes).

In one aspect, the present invention provides a sheet having an array structure formed by arranging a plurality of well units; a cell sheet having a two-dimensional structure attached to the side of the sheet having an opening in the well; and a patch for wound healing comprising cell spheroids having a three-dimensional structure existing inside the wells of the sheet.

In one embodiment, the cells are preferably human or mammalian cells, and may be fibroblasts, keratinocytes, cardiac fibroblasts, cardiomyocytes, stem cells or primary cultured cells.

In one embodiment, the sheet may be made of a biocompatible material, and the biocompatible material may be at least one selected from the group consisting of PDMS, silicone, polyethylene glycol, and alginate.

In one embodiment, the number of cells included in the patch may be 2x10 ⁵ to 5x10 ⁵ cell/cm ² .

In one embodiment, the patch may be used for wound treatment, and the two-dimensionally cultured cells and the three-dimensionally cultured cells can be directly delivered to a wound site at the same time without a cell collection step, thereby increasing cell delivery efficiency to the damaged site. can

In one embodiment, the wound is a burn, laceration, epidermal wound, ulcer, trauma, post-surgical, childbirth, chronic wound, damage due to dermatitis, corneal ulcer, corneal epithelium It may be any one or more selected from the group consisting of desquamation, keratitis, and damage due to dry eye syndrome.

In one embodiment, the patch may be for topical application to the skin.

In one aspect, the present invention manufactures a sheet of an array structure formed by aligning a plurality of well units with a biocompatible material; dispensing cells into sheets; It relates to a method for manufacturing a patch for wound healing, comprising culturing cells.

In one embodiment, a two-dimensional cell sheet may be prepared by two-dimensionally culturing cells on the side of a well having an opening.

In one embodiment, a three-dimensional cell spheroid may be prepared by culturing the cells in three dimensions inside the well.

In one embodiment, the cells are most preferably human or mammalian cells, and may be fibroblasts, keratinocytes, cardiac fibroblasts, cardiomyocytes, stem cells or primary cultured cells.

In one embodiment, the sheet may be made of a biocompatible material, and the biocompatible material may be at least one selected from the group consisting of PDMS, silicone, polyethylene glycol, and alginate.

In one embodiment, cells may be aliquoted and centrifuged at a speed of 400 g to 2000 g.

In one embodiment, cells may be cultured under hypoxic conditions.

In one embodiment, the patch may be used for wound healing.

In one aspect, the present invention manufactures a sheet of an array structure formed by aligning a plurality of well units with a biocompatible material; dispensing cells into sheets; A method for manufacturing a patch comprising a co-cultured structure of two-dimensional cells and three-dimensional cells, comprising culturing cells on the surface of a sheet having openings and inside the wells.

In one embodiment, the cells are most preferably human or mammalian cells, and may be fibroblasts, keratinocytes, cardiac fibroblasts, cardiomyocytes, stem cells or primary cultured cells.

In one embodiment, the sheet may be made of a biocompatible material, and the biocompatible material may be at least one selected from the group consisting of PDMS, silicone, polyethylene glycol, and alginate.

In one embodiment, cells may be cultured in two dimensions on the side of the well having openings to form a two-dimensional cell sheet, and cells may be cultured in three dimensions inside the well to form a three-dimensional cell spheroid.

In one embodiment, the fabrication process of the sheet includes lithography; etching techniques such as laser, plasma etching, photolithography, or chemical etching such as wet chemistry, drying, and photoresist removal; or by solid glass form techniques including three-dimensional printing (3DP), stereolithography (SLA), selective laser sintering (SLS), ballistic particle fabrication (BPM) and fused deposition modeling (FDM); by micromechanization; thermal oxidation of silicon; electroplating and electroless plating; diffusion processes such as boron, phosphorus, arsenic, and antimony diffusion; ion implantation; Film deposition, such as evaporation (filament, electron beam, flash, and shadowing and step coating), sputtering, chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, and molecular beam), electroplating, screen printing, lamination or by combinations thereof. See Jaeger, Introduction to Microelectronic Fabrication (Addison-Wesley Publishing Co., Reading Mass. 1988); Runyan, et al., Semiconductor Integrated Circuit Processing Technology (Addison-Wesley Publishing Co., Reading Mass. 1990); Proceedings of the IEEE Micro Electro Mechanical Systems Conference 1987-1998; See Rai-Choudhury, ed., Handbook of Microlithography, Micromachining & Microfabrication (SPIE Optical Engineering Press, Bellingham, Wash. 1997).

In one embodiment, the sheet serves as a scaffold for cells, and the skilled person may refer to resources available in the state of the art regarding the manufacture and use of tissue engineering scaffolds, in particular Dhandayuthapani et al. , “Polymeric Scaffolds in Tissue Engineering Application: A Review; International Journal of Polymer Science, Vol. 2011 (2011), pages 1-19. Shape of Scaffold Elements. , thickness, length, orientation, and surface topography characteristics allow the scaffold element to deform, bend, or otherwise change its shape in response to the action or activity of cells on its surface or within the well, and such deformation, bending, or other It can be varied in any number of suitable ways, as long as the change in shape can be measured reliably.

In one embodiment, the shape of the wells of the sheet of the present invention is not limited in any particular way, and may be square, rectangular, circular, oval, oval, triangular, or any combination of shapes. Other dimensions of the wells may also vary in any suitable way.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.

Example 1. CSCP fabrication

A co-spatial compartmentalized patch (CSCP) was fabricated using a patterned polyurethane-acrylate mold and 5 wt% polydimethylsiloxane (PDMS) (Sylgard, Dow Corning co., Midland, MI, USA). In addition, the negative tone photoresist SU-8 was micropatterned on the silicon wafer by a photolithography process. Specifically, a PDMS solution in which a PDMS base and a curing agent (Sylgard, Dow Corning Co.) were mixed at a ratio of 20:1 was poured into a custom mold, air bubbles were removed in a vacuum chamber, and cured in an oven at 80° C. for 2 hours. The cured PDMS replica is separated from the mold, and a well structure having a diameter within a known diameter (within 500 μm) of which 3D cells do not show toxicity is introduced on the PDMS plane by photolithography, and then a circular metal hole punch is used. A round-shaped patch CSCP with a diameter of 10 mm was fabricated by drilling a hole using the To observe the surface of the fabricated CSCP, the patch was placed in the sample chamber of a scanning electron microscope (SEM, JSM-6510, JEOL Ltd, Tokyo, Japan) and the surface was observed, and the depth of the hole made in the patch was measured using an optical microscope (CKX53, Olympus, Tokyo, Japan) and SEM were used to confirm and measure (Fig. 1a).

Example 2. 2D and 3D fibroblast culture in CSCP and check properties

After suspending the cells in a serum-containing medium, adding enough g that the cells do not interfere with viability due to the stress caused by the gravitational acceleration (g) of centrifugation (depending on the type of cell. Usually 400g to 2000g), It was cultured overnight for about 24 to 48 hours (or longer, depending on the cell). As an example of adherent cells, fibroblasts (human dermal fibroblasts) were each dispensed into CSCP and cultured for 24 hours under 5% CO ₂ conditions, and then 2D and 3D cultures of the cells were observed under a fluorescence microscope.

As a result, it was confirmed that a 2D structured cell sheet of fibroblasts was made on the outer surface of the CSCP, and 3D spheroids were made inside the well structure, and the spheroids made through the side cross section were also formed in a spherical shape. It was confirmed that it exists (Fig. 1b).

Example 3. 2D and 3D fibroblast co-culture CSCP in vitro Check wound healing effect

3-1. Confirmation of gene expression related to angiogenesis and cytokines in 2D and 3D fibroblasts of CSCP

In order to confirm whether the CSCP containing 2D and 3D co-cultured fibroblasts (FP) of the present invention has a wound healing effect through angiogenesis in the wound healing process, Angiogenic factors, pro-inflammatory cytokines and anti-inflammatory cytokine related genes were identified by qRT-PCR. Specifically, total ribonucleic acid from each of the 2D fibroblasts and 3D fibroblast groups (FP), the 2D fibroblasts alone group (Cell) and the 3D fibroblasts alone group (SP) adhered to and co-cultured with the CSCP of Example 2 ( RNA) was extracted with 1 mL TRIzol reagent (Life Technologies, Inc., Carlsbad, CA, USA) and 200 μL chloroform, and the RNA pellet obtained by centrifugation at 12,000 rpm for 10 min at 4 °C was obtained in 75% (v /v) washed with ethanol and dried. After drying, each sample was dissolved in RNase-free distilled water, and for qRT-PCR, SsoAdvanced ™ Universal SYBR Green Supermix kit (Bio-Rad, Hercules, CA, USA) and CFX Connect ™ real-time PCR detection system (Bio-Rad) were used. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), KLF-4 (Kruppel-like factor 4), E-CADHERIN, INVOLUCRIN, KI67, PCNA, HIF-1α, VEGF, CASPASE-3, BAX, BCL-2 and Involucrin The expression level was confirmed.

As a result, the expression of angiogenesis factor-related genes in 2D and 3D co-cultured fibroblasts (FP) on CSCP in Example 2 was higher than that of the 2D alone group and the 3D alone group (Cell and SP), and most of the inflammation In the case of related cytokines, there was no difference from the 2D alone group or showed median values (FIG. 2).

3-2. Confirmation of M2 macrophage polarization in 2D and 3D fibroblasts by CSCP

In order to confirm the effect of CSCP containing 2D and 3D co-cultured fibroblasts (FP) of the present invention on macrophage-initiated immune action during wound healing, 2D and 3D co-cultured fibroblasts (FP) cultured in CSCP and After culturing the cells of the same number of 2D fibroblasts alone group (Cell) and 3D fibroblasts only group (SP) in a serum-free medium for 24 hours, M2 polarization by various cytokines secreted into the medium was observed in M1 macrophages And it was confirmed by confirming the expression of M2 macrophage-related genes.

As a result, it was confirmed that M1-related markers decreased and M2-related markers increased in the FP group and the SP group (FIG. 3).

Example 4. 2D and 3D fibroblast co-culture CSCP in vivo Check wound healing effect

In order to confirm that the 2D and 3D fibroblasts supported on the CSCP prepared in Example 2 were actually delivered directly to the disease site in the preclinical stage, CSCP was prepared using dye-conjugated fibroblasts and transplanted into a wound-induced mouse After that, in vivo imaging was performed. Specifically, 8-week-old male athymic mice (weight 20-25 g, Dongyang, Seoul, Korea) were treated with 200 μL xylazine (20 mg/kg) and ketamine (100 mg/kg) diluted in normal saline. After anesthesia, two skin defects were marked using an 8 mm biopsy punch (LKF, Ansan, Korea), and the epidermis, dermis, and stratum corneum were surgically removed to induce wounds. After removing the skin, four 6-0 sutures (AILEE, Busan) were made at the border of each wound to prevent wound collapse due to skin contracture. Immediately after skin wound modeling, mice were treated with nothing (NT), CSCP group supported with 2D and 3D co-cultured fibroblasts (FP), 2D cultured fibroblasts alone group (Cell), and 3D cultured fibroblasts alone group (SP). ) and 2D and 3D cells from the CSCP patch and injected with a syringe (PDF). The FP group transplanted CSCP containing 2D and 3D co-cultured fibroblasts, the Cell group and the SP group and PDF group injected cells (direct injection method) into the muscle layer between diseased and normal tissue sites. All animals were managed according to the Guidelines for Care and Use of Laboratory Animals of Sungkyunkwan University (SKKUAICUC2020-01-12-1, January 2020). After that, to confirm the wound healing effect, the macroscopic wound area was quantified by processing pictures taken at different time points by tracing the wound margin and calculating the associated pixel area using a ruler, and the position of the healed wound edge was determined by the location of the wound. Centrally and defined as the macroscopic edge of epithelial migration over the granulation tissue bed. The wound area was calculated by Equation 1 below (Fig. 4A, B), and the quantitative average of fluorescence and photons was calculated using imaging software (ImageJ, NIH, MD, USA) using Lago-x (Spectral instrument imaging, USA). Morphometric analysis was performed on the digital images using As a result, dye signals were generally observed in the diseased areas of the FP group implanted with CSCP loaded with 2D and 3D co-cultured fibroblasts, and their quantitative values were significantly higher than those of the Cell, SP, and PDF groups (Fig. 4C to E).

In addition, as a result of staining the diseased area with DAPI and HNA antibody that showed a positive reaction to the nuclei of human cells, fibroblasts (hDF) transplanted in the group (FP) transplanted with the CSCP patch were also positive in the diseased section. A signal could be confirmed (Fig. 4F).

Example 5. 2D and 3D keratinocyte co-culture CSCP production and confirmation

5-1. CSCP fabrication and cell density confirmation

In order to confirm that 3D culture of cells that are difficult to culture by hanging drop due to lack of adhesive property is possible in CSCP, human keratinocytes were used to form 2D structured cells on the surface of CSCP. 2D and 3D keratinocyte co-culture CSCPs with 3D spheroids supported inside the sheet and well structures were fabricated. Then, 5 in keratinocyte growth medium 2 (PromoCell) supplemented with SupplementMix (PromoCell), CaCl ₂ stock solution (PromoCell) and 1% (v/v) penicillin/streptomycin (Gibco BRL, Gaithersburg, MD, USA). The cells were cultured under conditions of % CO ₂ saturation and 37° C., the medium was replaced every 2 days, and the cells were cultured for 48 hours under 5% CO ₂ conditions for cell culture after passage 4 times. After that, in order to investigate the cell density of the tissue culture plate (TCP) group using the fabricated 2D and 3D keratinocyte co-culture CSCP and existing tissue culture, the number of cells after trypsinization was calculated, and the following Equation 2 was used to calculate cell density based on dish and patch area. In addition, after scraping off the 2D keratinocytes growing in the CSCP, we tested the expression of apoptosis-related genes in 2D keratinocytes growing in the TCP and 3D keratinocyte clusters growing in the pores of the CSCP.

As a result, unlike the tissue culture plate (TCP) group, it was confirmed that the keratinocytes adhered well to the surface and inner well structures of the 2D and 3D keratinocyte co-cultured CSCPs and grew (FIG. 5B). In addition, the cell density of 2D and 3D keratinocyte co-cultured CSCP was found to be significantly higher than that of the TCP group (Fig. 5D), and in the case of the TCP group using the conventional 3D structure manufacturing method, the formation of a 3D structure was almost nonexistent. did not appear (Fig. 5E).

5-2. Identification of factors related to cell viability and angiogenesis in hypoxic conditions of keratinocyte 3D structures

To confirm the cell viability of keratinocytes cultured under hypoxic conditions, fluorescein diacetate (FDA, Sigma, St. Louis, MO, USA) (cytoplasmic staining of viable cells) and ethidium bromide (EB ) (nuclear staining of dead cells) (Sigma). The staining solution was prepared by mixing 10 μL of FDA stock solution (FDA 1.5 mg/mL in dimethyl sulfoxide) and 5 mL of EB stock solution (EB 1 mg/mL in phosphate buffered saline (PBS, Gibco BRL, Gaithersburg)) as described above. Cells of CSCP and TCP prepared in Example 5-1 were treated, respectively, and incubated at 37° C. for 3-5 minutes, and cell viability was observed under a fluorescence microscope (DFC 3000 G, Leica, Wetzlar, Germany). In addition, the CSCP and TCP cells were separated to confirm the expression levels of the cell proliferation-related factor PCNA, the cell death-related factor Caspase-3, and the angiogenesis factors HIF and VEGF, respectively.

As a result, more apoptotic cells (dead or dying cells with a red signal) were identified in the TCP group (FIG. 6). In the case of the 2D and 3D keratinocyte co-culture CSCPs of the present invention, both 2D and 3D cells were found to adhere well and survive. (Fig. 5E and F), it was found that the method of the present invention can implement 3D culture of cells with low adhesiveness, which is impossible in 3D cell culture by conventional methods. In addition, in the 2D and 3D keratinocyte co-culture CSCP of the present invention, PCNA, a factor related to cell proliferation, was decreased, but angiogenesis factors such as HIF and VEGF were greatly increased, and expression of apoptosis-related factors such as Caspase-3 was decreased. It was confirmed that it can be usefully used for wound healing by reducing (FIG. 7).

Claims (17)

A method of two-dimensionally and three-dimensionally co-cultivating cells on a sheet having an array structure formed by arranging a plurality of well units:
culturing the cells in two dimensions on the outer surface of the wells of the sheet; and
A method for culturing cells in three dimensions inside a well. The method of claim 1 , wherein the sheet is made of a biocompatible material. The method of claim 2, wherein the biocompatible material is at least one selected from the group consisting of PDMS (Polydimethylsiloxane), silicone (Silicone), polyethylene glycol (Polyethyleneglycol) and alginate (Alginate). a sheet having an array structure formed by arranging a plurality of well units;
a two-dimensionally structured cell sheet attached on the outer surface of the well of the sheet; and
A wound healing patch comprising cell spheroids having a three-dimensional structure existing inside a well of a sheet. The patch for wound healing according to claim 4, wherein the cells are fibroblasts or keratinocytes. The patch for wound healing according to claim 4, wherein the sheet is made of a biocompatible material. The patch for wound healing according to claim 6, wherein the biocompatible material is at least one selected from the group consisting of PDMS, silicone, polyethylene glycol and alginate. The patch for wound healing according to claim 4, which is used for wound healing. The patch for wound healing according to claim 4, comprising a co-culture structure of two-dimensional cells and three-dimensional cells. 1) fabricating a sheet having an array structure formed by aligning a plurality of well units with a biocompatible material;
2) dispensing cells into sheets;
3) A method for producing a patch for wound healing, comprising culturing cells. The method according to claim 10, wherein the two-dimensional cell sheet is prepared by culturing the cells in two dimensions on the outer surface of the well. According to claim 10, To prepare a three-dimensional cell spheroid by culturing the cells in three dimensions inside the well, the production method. The method of claim 10, wherein the biocompatible material is at least one selected from the group consisting of PDMS, silicone, polyethylene glycol, and alginate. The method according to claim 10, wherein the cells are divided and centrifuged at a speed of 400 g to 2000 g. delete The method of claim 10, wherein the cells are fibroblasts or keratinocytes. The manufacturing method according to claim 10, which is a patch for wound treatment.

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