Cultured Skin Substitutes Purity via Feeder Cell Inactivation
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Solution Overview
Problem
Current methods for developing cultured skin substitutes are limited by the fragility and susceptibility to infection of cultured skin autografts, which lack a dermal layer and contain residual feeder layer cells, leading to purity issues and high costs due to lengthy culture times.
Innovation Solution
The development of methods involving the use of a feeder layer with an agent that prevents cellular replication, such as mitomycin-C, followed by assaying and separating product cells from feeder layer cells to produce skin equivalents substantially free of residual feeder layer cells, using agents like EDTA for separation and PCR assays to ensure purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If feeder layer cells are used to support in vitro growth of keratinocytes, then cell growth is enhanced, but residual feeder layer cells contaminate the cultured skin substitutes reducing purity
Solution Approach 1:
The patent applies preliminary action by treating feeder layer cells with mitomycin-C before co-culturing with keratinocytes. This pre-treatment inactivates the feeder cells' replication capability while preserving their ability to support keratinocyte growth, thereby preventing contamination of the final product with proliferating feeder layer cells.
Solution Approach 2:
The patent employs extraction by removing the harmful property (replication capability) from the feeder layer cells through mitomycin-C treatment, while retaining the useful property (growth support function). This selective removal allows the feeder layer to fulfill its supportive role without contaminating the cultured skin substitutes.
2Ease of manufacture
If cultured skin autografts are used to treat wounded skin, then skin coverage is provided, but the grafts are fragile and susceptible to infection due to lack of dermal layer
Solution Approach 1:
The patent applies composite materials by combining keratinocytes with a dermal equivalent consisting of inactivated feeder layer cells and extracellular matrix components. This composite structure provides both the epidermal coverage function of keratinocytes and the structural support and protective functions of the dermal layer, resulting in more robust skin substitutes.
Solution Approach 2:
The patent employs the nested doll principle by placing keratinocytes on top of the dermal equivalent layer. The dermal equivalent serves as a foundational layer that supports and protects the overlying keratinocyte layer, creating a multi-layered structure that mimics natural skin architecture.
3Quantity of substance
If lengthy culture times are used to grow keratinocytes, then sufficient cell quantity is obtained, but production costs increase
Solution Approach 1:
The patent applies preliminary action by pre-expanding the keratinocyte population in co-culture with feeder layer cells before final product formulation. This pre-expansion phase allows rapid cell multiplication using the feeder layer's growth-promoting properties, reducing the overall culture time required to achieve sufficient cell quantities for therapeutic use.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach results in cultured skin substitutes that are more robust, less susceptible to infection, and significantly reduced residual feeder layer contamination, improving the purity and efficiency of the skin equivalent production process.
Implementation Method 1
providing product cells, feeder layer cells, and an agent that prevents cellular replication; treating the feeder layer cells with the agent that prevents cellular replication
Implementation Method 2
separating the product cells and the feeder layer cells; using agents like EDTA for separation
Implementation Method 3
assaying the product cells for the presence of feeder layer cell DNA; using PCR assays to ensure purity
Data Source
AI summary
The present invention relates to in vitro cultured skin substitutes. In particular, the present invention relates to compositions and methods for the development of cultured skin substitutes using NIKS cells.