WO2013191531A1

WO2013191531A1 - Autologous tissue-engineered human skin construct and a method for producing thereof

Info

Publication number: WO2013191531A1
Application number: PCT/MY2013/000112
Authority: WO
Inventors: Binti Idrus RUSZYMAH; Bin Saim AMINUDDIN
Original assignee: Universiti Kebangsaan Malaysia
Priority date: 2012-06-20
Filing date: 2013-06-19
Publication date: 2013-12-27
Also published as: MY179888A

Abstract

An autologous tissue-engineered skin construct for use in various dermatological therapies and clinical applications, comprising cultured-expanded human keratinocyte cells and human fibroblast cells originated from a collagenase-digested and trypsinized skin sample, wherein the human keratinocyte cells and human fibroblast cells has been co-cultured and subcultured respectively with human serum-incorporated medium.

Description

AUTOLOGOUS TISSUE-ENGINEERED HUMAN SKIN CONSTRUCT AND A METHOD FOR PRODUCING THEREOF

FIELD OF INVENTION

The present invention relates to culture-expanded human skin cells for the reconstruction of tissue-engineered skin, and a method for producing thereof. More particularly, the present invention provides autologous culture-expanded human fibroblast cells and human keratinocyte cells, as well as a technique for culturing thereof for obtaining a tissue-engineered skin construct or implant which could be used in various clinical applications.

BACKGROUND OF THE INVENTION

Skin is the largest organ in human body. Skin provides protection, sensation, thermoregulation and metabolic functions. There are two layers of tissue on the skin, namely the epidermis and dermis. Epidermis layer is made up of keratinocyte cells; whereas the dermis layer is made up of fibroblast cells. The keratinocyte cells will differentiate to become stratum corneum that later becomes the keratin layer at the surface of the skin. Whilst, the fibroblast cells secretes collagen, elastin and fibronectin. These are the main components of extracellular matrix that will provide strength to skin.

As skin forms such a large and essential part of human body, various techniques of skin tissue-engineering have been aggressively investigated by the researchers in recent years in order to provide a promising means for treating various dermatological symptoms and skin diseases. Apart from treating wound surface and replacing skin tissue lost, the tissue-engineered skin is also expected to be able to promote growth of skin, improve wound healing effect, prevent skin infections as well as reduce potential scarring of the skin.

In 1975, Rheinwald and Green started their work on in vitro culture of keratinocytes through combination of media containing fetal bovine serum (FBS), growth factor and feeder layer cells to produce cultured keratinocyte sheets (CKS) that can be transplanted to the patient. However, the persistence of feeder layer cells (xenogenetic mouse fibroblast) and proteins from the FBS used to grow CKS can cause late graft loss due to immunologic response. Therefore, an improved culture technique for producing a tissue-engineered skin construct has become one of the major interests in the research and development of skin reconstruction technologies.

There are a few patented technologies disclosed over the prior art relating to methods for culturing mammalian or human skin cells including the keratinocytes and fibroblasts for use in skin grafting and production of skin constructs. Different types of culturing techniques as well as the culture materials applied in the production of these tissue-engineered skin constructs could result in skin constructs of different qualities and applications. There is a formation of tissue-engineerd human skin using human plasma derivatives (HPD) as biomaterials disclosed in PCT publication No. WO2006001778. In this technology, an autologous bilayer human skin using human keratinocytes and dermal fibroblasts cultured from a patient sample is produced by mixing it with HPD. This technology requires separated steps of culturing keratinocytes and fibroblasts separately, before the cultured cells could be separately mixed with HPD to form the autologous bilayer of human skin. Therefore, the cell growth resulted from this technique is relatively slow. A more innovative technique which promotes faster cell growth and enhances the healing process is clearly desirable. Previous research on keratinocytes growth using serum-free medium and combination of growth factors has been shown to be able to produce purer keratinocytes culture. A U.K. Patent No. GB2394477 discloses a mammalian cell culture method and apparatus, in which a serum-free cell culture medium is applied. The cells are cultured using a culture vessel comprising a support with a surface of an acid monomer, an attached layer of fibroblast feeder cells and the serum-free culture medium. The culture cells could be keratinocytes, fibroblasts, skin stem cells, embryonic stem cells, melanocytes, corneal, intestinal mucosa, urethral, bladder, neuronal glial, hepatocyte stellate or epithelial cells. A co-culture technique is also disclosed, but a surface of acid monomer or copolymer, which is non-autologous, is required in this technique. There is no subculturing, specific cell separation or multiplication methods disclosed in this technology towards the production of a safe and complete tissue-engineered skin construct.

It was found that addition of fibroblast cells to the keratinocyte graft could also provide better outcome such as better skin graft uptake, quick healing and better cosmetics features. A U.S. Patent No. US2005019310 also discloses a method for culturing and expansion of mammalian undifferentiated human epidermal keratinocytes exhibiting stem cell characteristics. It could be co-cultured with human dermal fibroblasts utilizing a low calcium serum-free and animal by-product-free medium containing fibroblast growth factor (FGF) or a mimic thereof. However, there is also no technical guidance on the use of specific subculturing, cell separation and multiplication methods, nor specific digestive enzyme or human-based serum involved in this technology.

SUMMARY OF INVENTION

The primary object of the present invention is to provide a human-based autologous tissue-engineered skin construct for use in human skin reconstruction as well as treatment for various human dermatological symptoms and diseases. Another object of the present invention is to provide an efficient technique for co- culturing and subculturing human keratinocyte and fibroblast cells which promotes rapid cell growth.

Still another object of the present invention is to provide a technique for preparing a skin construct and a product of skin construct using an animal-free or xeno-free culturing system which is biocompatible to the human body and safe to be applied in various clinical applications.

Yet another object of the present invention is to develop a fast-healing process after skin reconstruction and thus preventing any delayed mobility and daily activities of the patients after the clinical operations or therapies. Further object another object of the present invention is to provide an autologous and culture-expanded human skin construct which could be directly used in the skin cell- or tissue-based therapies.

Another further object of the present invention is to develop an autologous tissue- engineered skin graft implantation for large area of skin defect considering the need of biocompatibility, dermal integration and mechanical strength in its design, and at the same time reducing the time, cost and risks involved.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention describes an autologous tissue-engineered skin construct for use in various dermatological therapies and clinical applications, comprising cultured-expanded human keratinocyte cells and human fibroblast cells originated from a collagenase-digested and trypsinized skin sample, wherein the human keratinocyte cells and human fibroblast cells has been co-cultured and subcultured respectively with human serum- incorporated medium.

In accordance with a preferred embodiment of the present invention, the skin sample is obtained autologously from a patient's own healthy skin. Preferably, the collagenase applied for digesting trypsinized skin sample is collagenase type 1; whereas the human serum applied is preferably 5 to 15%.

Another embodiment of the present invention is a method for culturing human keratinocyte cells and human fibroblast cells to obtain an autologous skin construct, comprising the steps of digesting a pretreated skin sample with collagenase to obtain digested skin cells; trypsinizing the collagenase-digested skin cells; culturing the trypsinized skin cells in a human serum-incorporated co-culture medium; separating the human fibroblast cells from the cultured skin cells upon cell confluence to be cultured separately in a human serum-incorporated culture medium; and subculturing the human fibroblast cells and the human keratinocyte cells respectively in another culture medium until a predefined number of cells is reached.

As set forth in the preceding embodiments, the skin sample is obtained autologously from patient's own healthy skin. The collagenase applied to digest the pretreated skin sample is preferably collagenase type 1.

According to the preferred embodiment of the present invention, the pretreated skin sample is obtained by cutting a skin sample into pieces, cleaning the cut skin sample from impurities and sterilizing the cleaned skin sample.

Still another preferred embodiment of the present invention discloses a method for culturing human keratinocyte cells and human fibroblast cells which further comprising a step of centrifuging and washing the trypsinized skin cells before the culturing step. In accordance with yet another embodiment of the present invention, the co-culture medium contains defined keratinocyte serum-free medium (DKSFM) and Dulbecco's modified eagle medium (DMEM); whereas the culture medium preferably contains DMEM. Both these co-culture medium and culture medium are incorporated with 5 to 15% of human serum.

The present invention discloses an innovative technology for culturing human dermal fibroblast cells and human keratinocyte cells from a skin biopsy. The present skin tissue-engineering technology invented is based on the co-culturing techniques, as well as the subculturing and multiplication of the cultured skin cells. Human serum is being used as opposed to FBS or any other types of animal-based serum in the existing technologies. Apart from that, the human fibroblast cells and human keratinocyte cells are cultured in an animal-free culture system which is capable of producing skin cells and tissue constructs that are safe and biocompatible to human body, thus preventing the occurrence of immunosupression in the patients. The human dermal fibroblast cells and human keratinocyte cells produced can be used for various clinical applications, including reconstruction of skin, treatment of dermatological symptoms and diseases, cell- and tissue-based therapies as well as drug testings. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to culture-expanded human skin cells for the reconstruction of tissue-engineered skin, and a method for producing thereof. More particularly, the present invention provides autologous culture-expanded human fibroblast cells and human keratinocyte cells, as well as a technique for culturing thereof for obtaining a tissue-engineered skin construct or implant which could be used in various clinical applications. Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The present invention discloses an autologous tissue-engineered skin construct for use • in various dermatological therapies and clinical applications, comprising cultured- expanded human keratinocyte cells and human fibroblast cells originated from a collagenase-digested and trypsinized skin sample, wherein the human keratinocyte cells and human fibroblast cells has been co-cultured and subcultured respectively with human serum-incorporated medium.

In accordance with a preferred embodiment of the present invention, the skin sample used for the production of the tissue-engineered skin construct is autologous, which obtained from a piece of patient's own healthy skin. This tissue-engineered skin construct or implant is cultured-expanded in an animal-free culturing system using non-animal digestive enzyme (trypsin), and serum derived from only human origin or autologous source.

Another embodiment of the present invention discloses a method for culturing human keratinocyte cells and human fibroblast cells to obtain an autologous skin construct, comprising the steps of digesting a pretreated skin sample with collagenase to obtain digested skin cells; trypsinizing the collagenase-digested skin cells; culturing the trypsinized skin cells in a human serum-incorporated co-culture medium; separating the human fibroblast cells from the cultured skin cells upon cell confluence to be cultured separately in a human serum-incorporated culture medium; and subculturing the human fibroblast cells and the human keratinocyte cells respectively in another culture medium until a predefined number of cells is reached.

The culturing technique of the human skin cells, including the human keratinocyte and fibroblast cells could significantly and directly affect the quality and properties of the engineered skin construct obtained. Therefore, intensive care shall be taken in each and every step of the culturing process in order to maintain the hygiene, purity as well as the viability of the culture skin cells and tissues.

According to the preferred embodiment of the present invention, the skin sample obtained autologously from the patient can be subjected to a pretreatment process as an initial step. The skin sample harvesting and processing method is detailed in Example 1. Accordingly, the skin sample is cut into pieces of appropriate sizes. The cut skin sample can then be cleaned from impurities including fat, hair and debris. Preferably, the pretreatment process also includes a sterilizing step in which the cleaned skin sample can be swab with alcohol; and then rinsed in a suitable buffered saline. In accordance with the preferred embodiment of the present invention, the buffered saline solution used is Dulbecco's phosphate buffered saline (DPBS).

The pretreated skin sample, which is preferably in pieces, can be further minced into a smaller size to ease the following digestion process that is performed using the collagenase. Preferably, the collagenase used in the present invention is Collagenase Type 1 which can be commercially obtained. The collagenase digestion process can be conducted in a incubator- shaker for 5 to 6 hours. The collagenase digestion is performed to break the peptide bonds in collagen in order to release the cells.

After completing the collagenase digestion, a non-animal-derived enzyme, which is trypsin, can be added to the collagenase-digested skin cells. The trypsin used is preferably a commercially available recombinant or synthetic trypsin. The trypsin treatment can be carried out in an incubator-shaker for 15 to 25 mins for keratinocyte cell dissociation. In accordance with still another preferred embodiment of the present invention, the method for culturing human keratinocyte cells and human fibroblast cells can further comprise a step of centrifuging and washing the trypsin-treated skin cells before the culturing step. Preferably, the cell suspension can be centrifuged at approximately 600x g for 5 min, and the pellet can be washed with DPBS.

Example 1 also shows an example of the co-culturing technique of the skin cells. The pellet containing the skin cells can be resuspended in the co-culture medium. In accordance with yet another embodiment of the present invention, the co-culture medium contains DKSFM and DMEM. Preferably, the co-culture medium used is DKSFM and F12: DMEM, which is incorporated with 5% to 15%, more preferably, 10% human serum is applied in the co-culture medium. The human serum is preferably used to culture the cells as it secrets a number of growth factors which promotes the cell growth. The cells can be cultured in suitable cell culture plate, supplemented with approximately 5% of carbon dioxide. Medium can be changed periodically, preferably every 2 to 3 days.

According to the preferred embodiment of the present invention, the fibroblast cells can be removed and separated from the keratinocyte cells in the cell culture when the cells multiply and reach confluence. Subsequently, the fibroblast cells are cultured separately to allow the further expansion and multiplication of cells. The culture medium used can be DMEM, preferably F12: DMEM, which is also incorporated with 5% to 15%, preferably 10% of human serum.

In order to promote the cell growths, the keratinocytes and fibroblasts can then be subcultured respectively after reaching 70% of cell confluence. As set forth in the preceding description, the recombinant or synthetic trypsin can be added into the respective subculture of keratinocytes and fibroblasts with predetermined seeding density. The cells are continuously subcultured until a predefined number of cells is reached in order to obtain the tissue-engineered skin construct. A exemplary differential trypsinization for fibroblast and keratinocyte culture separation is further detailed in Example 2.

Appropriate tests including shelf-life evaluation of the skin construct at different storage time, histological analysis, immunocytochemistry and immunohistochemistry studies can be carried out to experiment the quality of the skin construct invented. The cell viability and growth kinetics after storage as well as the quantitative gene expression analysis of the skin construct by real-time polymerization chain reaction (PCR) can also be conducted. The human keratinocyte cells and human fibroblast cells can be cultured on a biocompatible medical silk which could be commercially obtained, in order to provide a physical support to the skin construct. The skin construct and the silk can be sewn together onto the patient's skin during the skin cell therapy or surgery, and the silk would automatically detached from the skin during the healing process. The constructed skin tissues can be preshaped according to the skin defect area of the patient. It can also be fabricated into a standard dimension which is to be cut by the surgeon during the operations. The bilayer skin construction is further detailed in Example 3. The skin construct can be integrated completely with the patient's skin after grafting, thus is expected to have comparatively faster wound closer. The stored skin construct does not show any significant change in the histological features and morphological property. Moreover, cells liberated from the skin construct which is stored at different time periods are able to maintain viability of above 90% and retain their proliferative potential, showing that the skin construct can be stored for at least 72 hours prior to grafting without significantly compromising the quality of the construct, yet exhibiting population doubling time. The cells in the construct are also distributed homogeneously, which allows ample space for cell proliferation when grafted onto patient. The long shelf-life of the skin construct also enables this skin substitute to be transported to other regions of the country and even to other parts of the world when maintained under good storage conditions.

The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

EXAMPLE

Examples are provided below to illustrate different aspects and embodiments of the present invention. These examples are not intended in any way to limit the disclosed invention, which is limited only by the claims.

Example 1

Skin samples from donors (patients) undergoing abdominoplasty or face-lift were obtained with informed consent. The skin tissues were processed within 24 hours from harvest. Blood was also collected from the same donors, from which the human serum was isolated from the human patient and stored for use as growth supplement in the culture media. Skin samples were cut into pieces, preferably in a standard size of 1mm x 2mm, and then was cleaned from fat, hair and debris. The skin was swab with 70% isopropyl alcohol and then rinsed with calcium and magnesium-free DPBS. The skin was minced to smaller size and was digested with Collagenase Type 1 in a incubator- shaker for 5 to 6 hours at 37°C. After complete digestion with Collagenase Type 1, a non-animal-based recombinant trypsin enzyme (TrypLE Select) was added to the pellet and incubate in an incubator-shaker for 20 min at 37°C for keratinocyte cell dissociation to obtain both keratinocytes and dermal fibroblasts. The cell suspension was centrifuged at 600 x g for 5 min and the pellet was washed with DPBS. Cell pellet were then resuspended in co-culture medium DKSFM and Nutrient Mixture F12: DMEM with 10% human serum and were cultured in 3 wells of six-well plate at 37°C in 5% CO2 with medium change every 2-3 days. Example 2

When the co-culture has reached 70 to 80% confluency, the culture medium was removed and the cells were rinsed with DPBS. Following this, 2 ml of TrypLE Select was added into each well and incubated for 5 minutes in a 37 °C incubator with 5% C0₂ to detach only the fibroblasts. The fibroblasts were collected and then cultured in T75 flask with F12: DMEM + 10% human serum and the medium was changed every 48 to 72 hours. The remaining keratinocytes still attached on the plate were rinsed with DPBS and continued to be cultured in DKSFM with medium change every 48 to 72 hours. Subsequently, upon reaching 70 to 80% of confluency, keratinocytes and fibroblasts were trypsinized with TrypLE Select and subcultured with a seeding density of 1.0 x 10⁵ cells per well (keratinocytes) or flask (fibroblasts) in their respective culture media until the desired amount of cells were obtained for the formation of the bilayered skin construct.

Example 3

A piece of surgical silk (Boston Medical Products, USA) was cut to the size of 9.6 cm² and placed into one well of a six-well culture plate under sterile condition. The silk was affixed to the well with a few drops of 10% CaCl₂ (Calcium Chloride Dihydrate, American Regent, USA). Any excess CaCl₂ was then removed from the well. The keratinocytes were trypsinized and cell count was performed by trypan blue staining to ensure sufficient amount of cells were acquired (l-2xl0⁶ cells). The cell suspension was centrifuged at 700xg for 5 minutes and pellet was then suspended with 2ml of the donor' s autologous plasma and 10% CaCl₂ was added to the mixture to initiate the polymerization process. The admixture was then dispensed quickly into well, on top of the silk, for polymerization to take place in order to form the fibrin- keratinocyte layer. The fibroblasts were trysinized and cell count was performed by trypan blue staining to ensure sufficient amount of cells we acquired (l-2xl0⁶ cells). The cell suspension was centrifuged at 700xg for 5 minutes and the pellet was then suspended with 1ml of the donor's autologous plasma and 10% CaC was added to the plasma mixture to initiate the polymerization process. This admixture was quickly laid on top of the fibrin-keratinocyte layer to form the fibrin-fibroblast layer and complete the final bilayered construct. The bilayered construct was immersed in 2ml of basal F12:DMEM medium (1: 1 ration without human serum) in the culture plant, sealed with parafilm and stored at 4°C. Humidity control is not necessary. The morphology of the fibroblasts remained spindled-shaped and the keratinocyte colonies appeared as groups of polygonal cells.

Claims

1. An autologous tissue-engineered skin construct for use in various dermatological therapies and clinical applications, comprising cultured- expanded human keratinocyte cells and human fibroblast cells originated from a collagenase-digested and trypsinized skin sample, wherein the human keratinocyte cells and human fibroblast cells has been co-cultured and subcultured respectively with human serum-incorporated medium.

2. A skin construct according to claim 1, wherein the skin sample is obtained autologously from a patient's own healthy skin.

3. A skin construct according to claim 1, wherein the collagenase applied is collagenase type 1.

4. A skin construct according to claim 1, wherein the human serum applied is 5 to 15%.

5. A method for culturing human keratinocyte cells and human fibroblast cells to obtain an autologous tissue-engineered skin construct, comprising:

digesting a pretreated skin sample with collagenase to obtain digested skin cells;

trypsinizing the collagenase-digested skin cells;

culturing the trypsinized skin cells in a human serum-incorporated co-culture medium;

separating the human fibroblast cells from the cultured skin cells upon cell confluence to be cultured separately in a human serum-incorporated culture medium; and

subculturing the human fibroblast cells and the human keratinocyte cells respectively in another culture medium until a predefined number of cells is reached.

6. A method according to claim 5, wherein the skin sample is obtained autologously from a patient's own healthy skin.

7. A method according to claim 5, wherein the pretreated skin sample is obtained by cutting a skin sample into pieces, cleaning the cut skin sample from impurities and sterilizing the cleaned skin sample.

8. A method according to claim 5, wherein the collagenase applied is collagenase type 1.

9. A method according to claim 5 further comprising a step of centrifuging and washing the trypsin-treated skin cells before the culturing step.

10. A method according to claim 5, wherein the co-culture medium contains defined keratinocyte serum-free medium and Dulbecco's modified eagle medium.

11. A method according to claim 5, wherein the culture medium contains Dulbecco's modified eagle medium.

12. A method according to claim 5, wherein the human serum incorporated in the co-culture medium and the culture medium is 5 to 15%.