Live-cell seeding method for microarrays
Inventors
Messner, Jacob • Anderson, Clifford • Glenn, Honor • Lee, Kristen • Richards, Mark • Kelbauskas, Laimonas • Bussey, Kimberly • Meldrum, Deirdre
Assignees
Arizona State University ASU • Arizona State University Downtown Phoenix campus
Publication Number
US-11136614-B2
Publication Date
2021-10-05
Expiration Date
2036-10-07
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Methods for seeding live cells onto spatially defined regions of a substrate including multiple features (e.g., microwells or other microenvironments) utilize a stencil embodied in a hole-defining sacrificial film. A sacrificial film devoid of holes may be applied over features of a substrate, and a hole generating mechanism (e.g., hot needle or laser) aligned with features may be used to define holes in the film. Alternatively, holes may be predefined in a sacrificial film to form a stencil, and the stencil may be assembled to the substrate with the holes registered with features thereof. Thereafter, cells are seeded through holes in the film. Seeded cells are subject to incubation, further processing, and/or performance of one or more assays, and the hole-defining sacrificial film (stencil) may be removed.
Core Innovation
This invention provides methods for seeding live cells onto spatially defined regions of a substrate, particularly onto features such as microwells or other microenvironments, by employing a stencil made from a sacrificial, typically biocompatible polymeric, film. The method can involve either in situ fabrication of holes in a film after it is affixed to the substrate or the use of a pre-perforated stencil assembled to the substrate so that its holes are aligned with defined features like microwells. Live cells are then seeded through these holes, ensuring precise spatial localization of the cells.
The invention addresses the drawbacks of conventional cell seeding methods. Traditional random seeding leads to undesirable cell adhesion on non-target surfaces such as microwell lips and interstitial areas, complicating downstream analysis and reducing single-cell isolation efficiency. Other approaches, like using adhesion-promoting chemistries or microfluidic sorting, introduce biases, stress, or require complex equipment, all of which are significant limitations for high-throughput single-cell analyses or metabolic assays.
By utilizing a precisely aligned stencil with holes registered to substrate features, this method minimizes undesired cell presence outside the target regions. Optionally, the stencil may be removed after cell seeding, leaving only spatially localized cells. The technique is compatible with standard array processing and further assays (such as metabolic flux measurement), and is specifically designed to be robust, simple, scalable, and compatible with high single-cell occupancy while avoiding stress or phenotypic bias in the cell population.
Claims Coverage
The patent contains two independent claims, each directed to core inventive features for methods of seeding cells using a sacrificial film stencil aligned to microwells.
Method for seeding cells using a sacrificial polymeric film stencil with in situ hole formation and alignment to microwells
This feature consists of: - Affixing a sacrificial polymeric film to a cell seeding substrate with a plurality of microwells, each being elevated or recessed relative to a connecting body structure. - Selectively melting the polymeric film to a cover in multiple locations to locally adhere specific portions. - Generating an array of holes by removing the cover, so that adhered portions remain on the cover and the sacrificial film on the substrate has holes registered with the microwells. - Seeding cells through these holes to achieve spatially localized cell placement. - Removing the sacrificial film after cell seeding. - Ensuring each hole in the film is smaller than the maximum width of each microwell.
Method for seeding cells using a sacrificial polymeric film selectively melted to a cover to produce registered holes for controlled cell deposition
This inventive feature includes: - Affixing a polymeric sacrificial film to a substrate defining microwells by selectively melting portions onto a cover, locally compromising structural integrity and causing adherence in multiple locations. - Generating a registered array of holes by removing the cover, resulting in holes aligned with microwells. - Seeding cells through the holes onto the substrate for spatially localized cell placement. - Removing the sacrificial film from the substrate after seeding. The method ensures spatial localization by aligning the holes with substrate features and includes the step of selectively compromising the mechanical integrity of the film via melting for precise removal and hole formation.
The inventive features focus on the use of a sacrificial polymeric (or optionally metal) film as a stencil, hole creation via selective melting and cover removal to register holes precisely with microwell positions, and subsequent methods of seeding and localizing live cells for downstream applications.
Stated Advantages
The method is robust, simple, and high-throughput, allowing for efficient and selective cell seeding onto spatially defined regions such as microwells.
Reduces the presence of cells on microarray lips and in interstitial areas, minimizing unwanted adherence and simplifying downstream analysis.
Helps avoid the crushing of cells and enables microarray devices to be suitable for metabolic assays by minimizing non-specific cell presence.
Enables high single-cell occupancy rates while using standard cell culture techniques, without requiring specialized equipment or expertise.
Does not expose cells to physical stresses, phenotypic selection, or environmental factors that may alter cellular physiology, maintaining cell health and population representation.
Stencil preparation is simple, low-cost, and accessible, with no need for cleanroom processing or complex alignment procedures.
Documented Applications
Single cell analysis on microwell arrays or other spatially defined microenvironments.
Metabolic flux assays involving measurement of oxygen concentration data.
Cell localization for tissue engineering, cell signaling studies, drug screening, and cell migration assays.
Interested in licensing this patent?