Stem cells have revolutionized the field of regenerative medicine, offering promising solutions for various medical conditions, including difficult-to-heal skin wounds. This review focuses on the background and manufacturing processes of skin cell-based therapies, particularly keratinocytes and adipose-derived mesenchymal stem cells (AD-MSCs), as highlighted in the thesis by Hady Shahin[1]. This article provides an overview of services offered by Contract Development and Manufacturing Organizations (CDMOs) involved in cell-based solution production.
Stem cells have unique abilities to self-renew and to recreate functional tissues. They can develop into many different cell types in the body during early life and growth[2]. Researchers study many different types of stem cells, including pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) and non-embryonic or somatic stem cells (commonly called adult stem cells)[2]. Pluripotent stem cells have the ability to differentiate into cells of the 3 main germ layers in the adult body[2]. Adult stem cells are found in specific anatomical locations and can differentiate to yield the specialized cell types of that tissue or organ[2]. They serve as an internal repair system that generates replacements for cells lost through normal wear and tear, injury, or disease.
Stem cells have the remarkable potential to renew themselves and to differentiate into various specialized cell types[2]. When a stem cell divides, the resulting two daughter cells may be both stem cells, a stem cell and a more differentiated cell, or both more differentiated cells[2]. Discovering the mechanism behind self-renewal may make it possible to understand how cell fate is regulated during normal embryonic development and post-natally, or mis-regulated during aging or in the development of cancer[2].
The skin, the largest organ of the body, serves as a protective barrier against the external environment. It consists of three distinct layers: the epidermis, dermis, and subcutaneous adipose tissue[1]. The epidermis, primarily composed of keratinocytes, plays a crucial role in maintaining skin integrity and facilitating wound healing. Keratinocytes move from the basal layer to the surface, undergoing differentiation and forming a protective barrier[1].
Difficult-to-heal wounds, such as those caused by chronic diseases, trauma, or burns, pose significant challenges in clinical practice. These wounds often result in prolonged pain, infection, and impaired quality of life[1]. Regenerative advanced therapy medicinal products (ATMPs), including cell-based approaches, offer promising solutions for enhancing wound healing and improving patient outcomes[1].
Keratinocytes —the most abundant cell type in the epidermis— are instrumental in the re-epithelialization process during wound healing. They proliferate and migrate to cover the wound bed, forming new epidermal layers[1]. An autologous therapeutic approach involves harvesting skin biopsies from a patient’s healthy donor sites, isolating keratinocytes, expanding them, and reapplying them to the same patient after thorough characterization, quality control, and safety testing. The classical method for culturing keratinocytes includes enzymatic digestion of the epidermis, followed by expansion in culture media [1]. However, a major challenge in this process is the use of animal-derived products, which poses regulatory hurdles [1]. To address these challenges, Shahin’s thesis proposes a xeno-free workflow for keratinocyte isolation and expansion. The study validates the use of a xeno-free workflow to manufacture human keratinocytes as ATMP [1]. This approach ensures the production of keratinocytes that comply with regulatory standards, making them suitable for clinical applications [1].
The allogeneic approach, on the other hand, involves Adipose-Derived Mesenchymal Stem Cells (AD-MSCs) as a promising alternative to address the scalability challenges associated with keratinocytes, which are mature cells. AD-MSCs are multipotent stem cells isolated from adipose (fat) tissue. They possess the ability to differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes[1]. AD-MSCs are particularly attractive for regenerative therapies due to their ease of isolation, high yield, and immunomodulatory properties[1].
In the context of wound healing, AD-MSCs contribute to tissue repair by promoting angiogenesis, reducing inflammation, and enhancing collagen synthesis[1]. Shahin’s thesis explores the potential of AD-MSCs as an alternative to keratinocytes for treating difficult-to-heal wounds[1]. The study highlights the differentiation of AD-MSCs into keratinocyte-like cells through direct co-culture with keratinocytes[1]. This approach leverages the paracrine signaling between the two cell types to enhance the differentiation process[1].
The manufacturing of stem cells for clinical applications involves several critical steps, including cell isolation, expansion, and quality control. Ensuring compliance with Good Manufacturing Practice (GMP) guidelines is essential to produce safe and effective cell-based therapies[1].
Every cell therapy product is special, NorthX Biologics adopts a flexible manufacturing operation where we together with the client tailor a manufacturing and testing process meeting the specific product’s exact requirements.
Keratinocytes are typically isolated from skin biopsies using enzymatic digestion. Shahin’s thesis validates the use of a completely xeno-free keratinocytes extraction method, ensuring the production of GMP-compliant keratinocytes in a timely manner[1]. AD-MSCs are isolated from adipose tissue through enzymatic digestion and centrifugation[1]. The high yield of AD-MSCs from adipose tissue makes them a viable option for large-scale production[1].
The expansion of keratinocytes and AD-MSCs requires optimized culture conditions to maintain cell viability and functionality. Shahin’s study demonstrates the use of xeno-free culture media for keratinocyte expansion, eliminating the need for animal-derived products[1]. For AD-MSCs, the co-culture with keratinocytes enhances their differentiation into keratinocyte-like cells, providing a scalable approach for producing epidermal cells [1].. Rigorous quality control is needed to ensure such in-vitro cell manipulation is safe and does not compromise the properties of the cells. Therefore, thorough characterization and stability testing are needed for cell-therapies to be considered safe for clinical use and to fulfil stringent regulatory requirements for ATMPs.
At NorthX Biologics, we provide an extensive range of testing services tailored to the advanced products we manufacture, to support designing a comprehensive analytics panel for your ATMP (consulting on release criteria with the regulatory bodies).
As cell therapy manufacturing for clinical use must be conducted under strict control in a GMP facility, the final cell solution often needs to be transported from the production site to the treatment site, which may be several hours away. Shahin’s study demonstrated that the cell solution can be transported for up to 24 hours under controlled conditions while maintaining cell functionality and characteristics.
This finding allowed the research team to establish human serum albumin as the preferred carrier solution for keratinocytes in clinical treatments, ensuring their viability and functionality during transport. Additionally, it was validated as the final formulation solution for administration.
At NorthX Biologics, we offer tailored fill & finish solutions with integrated analytical support.
Ensuring the quality and safety of cell-based ATMPs is paramount. Shahin’s thesis emphasizes the importance of thorough characterization of keratinocytes and AD-MSCs, including the assessment of cell viability, differentiation potential, and functionality[1]. The use of cell and molecular characterization methods, including but not limited to immunophenotyping, gene and protein expression analyses are instrumental tools for monitoring and ensuring the quality of the produced cells [1].
The thesis by Hady Shahin offers valuable insights into manufacturing cell-based regenerative solutions for skin healing. The proposed xeno-free workflows and co-culture techniques present promising methods for producing GMP-compliant cell therapies. These advancements pave the way for effective treatments for difficult-to-heal wounds, ultimately improving patient outcomes.
Hady Shahin, PhD
Production Scientist
NorthX Biologics
Hady is a Production Scientist at NorthX Biologics, providing bioprocess and CMC support for GMP manufacturing of drug substances (DS), contributing to the production of advanced biologics for clinical trials. Biotechnologist by training and SME in scaling up cell therapy solutions, with over 15 years of experience in regenerative medicine and stem cell research. Hady holds a PhD in Cell and Molecular Biology and an MSc in Biomedical Science, with a strong background in ATMP development and GMP manufacturing.
NorthX Biologics is a CDMO and Innovation Hub in Advanced Biologics, with +30 years of GMP production experience. The team provides process development and GMP manufacturing services with expertise in plasmid DNA, mRNA, proteins, cells and other advanced biologics. Headquartered in the heart of Sweden, the team serves customers worldwide and in 2021 was recognized as a national innovation hub for advanced therapeutics and vaccines. NorthX Biologics has the ambition to become a leading cell and gene therapy manufacturer and partner of choice for innovative drug development companies.
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