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-9243223-B2
Publication Date
2016-01-26
Expiration Date
2030-09-13
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Abstract
A model of tissue is produced by steps comprising 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 cells that have not partially attached, 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 muscle tissue or other tissue depending on the cells seeded.
Core Innovation
The invention relates to methods for culturing cells to form tissue models, particularly models of muscle tissue, through a process of high-density spot seeding of progenitor cells on a support. Cells are seeded at a selected concentration to form a cell spot, incubated to allow partial attachment, gently rinsed to remove unattached cells, and then cultured in growth medium to induce proliferation primarily at the periphery of the cell spot while cells toward the center differentiate. This results in cell alignment, fusion, and maturation into functional tissue models such as muscle tissue.
The problem being addressed is the difficulty of producing well-aligned, spontaneously contracting muscle tissue models without resorting to specialized substrates, costly differentiation media, or complex scaffolding, which are known in the related art. The disclosed method provides a simple, reproducible, and cost-effective way to form two-dimensional or three-dimensional tissue models that mimic natural muscle tissue alignment and differentiation through relying on cellular mechanisms such as contact inhibition and spatially controlled cell proliferation.
Claims Coverage
This patent includes one independent claim with multiple inventive features related to producing a three-dimensional muscle tissue model via a multi-step cell culturing process.
High-density cell spot seeding with specific cell concentration and volume
Seeding anchorage-dependent cells onto a support at a concentration ranging from about 1×10^5 to about 1×10^6 cells per milliliter, with each spot volume between about 20 µl to about 50 µl, forming a cell spot with cells inducible into muscle cells.
Selective partial cell attachment and removal of unattached cells
Incubating cells for an initial period to allow partial attachment to the support, followed by removal of any cells that have not partially attached.
Use of growth medium volume that supports undisturbed proliferation without refresh for at least seven days
Adding a volume of growth culture medium sufficient to enable the partially attached cells to proliferate about the periphery and differentiate away from the center, allowing undisturbed cell growth and maturation for a period of at least seven days without medium refresh.
Formation of functional muscle tissue layers by further incubation
Further incubating the cells to cause alignment, fusion, and maturation into a first layer of functional muscle tissue.
Layered assembly of three-dimensional tissue model
Producing a second muscle tissue layer in the same manner and positioning it on top of the first layer with an extracellular matrix component layer in between to create a three-dimensional tissue model.
The claims collectively cover a process of forming a three-dimensional muscle tissue model by precise high-density spot seeding, partial attachment with removal of unattached cells, controlled undisturbed cell proliferation and differentiation, and multilayer tissue assembly with interposed extracellular matrix components.
Stated Advantages
Provides a simple, reproducible, and cost-effective method to produce aligned, spontaneously contracting muscle tissue models without requiring specialized substrates or differentiation media.
Enables formation of two-dimensional or three-dimensional tissue models that mimic natural muscle tissue alignment and function.
Facilitates understanding of skeletal muscle maturation and muscle-related disorders such as muscle atrophy.
Allows potential applications in treatment or repair of muscle damage including muscle atrophy, trauma, and cardiac infarction.
Can serve as powerful tools to elucidate molecular mechanisms of cell differentiation and muscle contraction when combined with other cell culture techniques.
Documented Applications
Models for studying skeletal muscle maturation processes including proliferation, alignment, fusion, differentiation, and contraction.
Biomedical and regenerative medicine applications such as tissue patching or repair for bone, skeletal muscle, and cardiac muscle.
Formation of three-dimensional tissue constructs by laminating monolayers with extracellular matrix components for regenerative medicine.
Creation of integrated tissue model systems combining muscle tissue with nerve or blood vessel models to study interacting tissues.
Use in elucidating molecular events of muscle atrophy and testing interventions to prevent or alleviate muscle atrophy.
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