High-density spot seeding for tissue model formation
Inventors
Marquette, Michele L. • Sognier, Marguerite A.
Assignees
National Aeronautics and Space Administration NASA
Publication Number
US-8735116-B2
Publication Date
2014-05-27
Expiration Date
2030-09-13
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Abstract
A method for making a tissue includes seeding cells at a selected concentration on a support to form a cell spot, incubating the cells to allow the cells to partially attach, rinsing the cells to remove any unattached cells, adding culture medium to enable the cells to proliferate at a periphery of the cell spot and to differentiate toward a center of the cell spot, and further incubating the cells to form the tissue. The cells may be C2C12 cells or other subclones of the C2 cell line, H9c2(2-1) cells, L6 cells, L8 cells, QM7 cells, Sol8 cells, G-7 cells, G-8 cells, other myoblast cells, cells from other tissues, or stem cells. The selected concentration is in a range from about 1×105 cells/ml to about 1×106 cells/ml. The tissue formed may be a skeletal muscle tissue, a cardiac muscle tissue, nerve tissue, or a bone tissue.
Core Innovation
The invention relates to a method for culturing cells to form tissue models, particularly models of muscle tissue, using high-density spot seeding of progenitor cells on supports such as tissue culture plates. Cells are seeded at concentrations ranging from about 1×10^5 cells/ml to about 1×10^6 cells/ml to form a cell spot, incubated to allow partial attachment, gently rinsed to remove unattached cells, and then incubated further with growth medium to enable cells to proliferate at the periphery and differentiate toward the center, ultimately forming functional tissue.
The method can employ various cell types including myoblasts like C2C12 cells and other subclones, stem cells, or de-differentiated cells. The resulting tissue may be skeletal muscle, cardiac muscle, nerve, or bone tissue. This approach leverages contact inhibition to create a differentiation timeline within the cell spot, with proliferating cells at the periphery and differentiating cells at the center, promoting alignment, fusion, and maturation into tissue models without requiring special substrates or differentiation media.
The problem solved is the lack of simple, reproducible, and cost-effective methods to produce aligned, contracting muscle tissue models in vitro. Previous methods failed to generate well-aligned myotubes or required costly substrates and media. This invention provides a high-density seeding technique that induces natural cellular behaviors and alignment, mimicking native muscle tissue structure and function, thereby enabling tissue formation suitable for biomedical research and potential regenerative medicine applications.
Claims Coverage
The patent contains one main independent claim defining a method for making a model of aligned, contracting muscle tissue. The following inventive features are described in the independent claim.
High-density cell spot formation
Seeding anchorage-dependent cells onto a support to form a cell spot at a concentration between about 1×10^5 and 1×10^6 cells per milliliter with each spot volume ranging from about 20 to 50 microliters.
Partial cell attachment and removal of unattached cells
Incubating cells for an initial time to allow partial attachment followed by removing any cells that have not attached after this initial period.
Proliferation at the cell spot periphery and differentiation toward the center
Adding growth culture medium after removal of unattached cells to enable the partially attached cells to proliferate at the periphery of the cell spot and differentiate away from the center.
Undisturbed culture period with sufficient medium volume
Using a volume of growth culture medium sufficient to allow the cells to proliferate undisturbed without a medium refresh for at least seven days, followed by further incubation to form the muscle tissue model.
The independent claim covers a method that integrates high-density spot seeding with partial attachment washing and controlled culture conditions to produce aligned, contracting muscle tissue models that exhibit differentiation and proliferation spatial organization.
Stated Advantages
Simple, reproducible, and cost-effective method for producing two-dimensional or three-dimensional tissue models.
Capability to form aligned, spontaneously contracting myotubes without the use of special media or costly substrates.
Mimics natural muscle tissue alignment and function more effectively than other known methods such as serum induction or nanofiber scaffolding.
Enables formation of tissue models useful for studying muscle maturation, atrophy, and contraction mechanisms and for biomedical and regenerative medicine applications.
Documented Applications
Use in forming muscle tissue models to study skeletal muscle maturation, including proliferation, alignment, fusion, and differentiation.
Application in understanding mechanisms of muscle atrophy and developing methods to alleviate or prevent muscle atrophy.
Treatment or repair of muscle damage, such as in muscle atrophy, skeletal muscle trauma, or muscle damage from cardiac infarction.
Production of small tissue pieces engineered for regenerative medicine, including tissue patching or repair using transplantable bone, skeletal muscle, or cardiac muscle tissues.
Combining with gene silencing or gene expression modulation techniques to elucidate molecular events in muscle differentiation and contraction.
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