You are here

Alternatives to commercially available cell culture insert membranes and manufacturing techniques


Fast-Track proposals will not be accepted. Direct-to-Phase II proposals will not be accepted. Number of anticipated awards: 1-3 Budget (total costs, per award): Phase I: $225,000 for 9 months; Phase II: $1,500,000 for 2 years PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED. Phase II information is provided only for informational purposes to assist Phase I offerors with their long-term strategic planning. Summary There has been an increased focus in the life sciences industry on the use of more complex 3D cellular and tissue models to help provide physiologically relevant platforms to be used in in-vitro drug testing. Nearly all high throughput in-vitro drug testing experiments utilize multi-well microtiter plates to act as a vessel where individual reactions between the biological model and the sample under test occur. The use of these more complex 3D cellular models has driven the use of more complex microtiter plates, namely cell culture insert-plates (CCIP) with permeable membranes. There are several factors that make these CCIPs suitable for use in the creation of complex cellular models, including the ability to co-culture cells with or without cell-to-cell contact, allowing for either apical or basolateral feeding to promote cellular metabolic activities, and provide an anchorage point which is typically required for most mammalian cells to remain viable. All these factors are needed to support cell proliferation and function, as well as formation of complex tissues, making the choice of membrane available for use in CCIPs a critical one towards the production of a physiologically relevant tissue model. The current membranes used in CCIPs are made using a variety of materials with options in pore size, coatings and surface treatments that can be selected depending upon the type of cell model to be produced. Most of these models incorporate the use of biomaterials to be used as scaffolds for the cells to be functional and create a new tissue. Many of the biomaterials used are naturally occurring and readily available such as collagen, alginate, chitosan and others. In many cases, these biomaterials are present in the extracellular matrix (ECM) produced by cells, so their use helps to provide a natural environment for the cells until they are healthy and producing ECM of their own. Aside from the biocompatibility of these scaffolding biomaterials, another factor is controlled biodegradability. This rate of biodegradation is a key factor for certain tissue model types where an initial scaffold is necessary to promote structural integrity, cellular anchorage, viability and proliferation but where over time the desire is for the scaffold to degrade and be replaced by naturally produced ECM and promote cell to cell interaction in co-culture models. The need for biodegradation of certain tissue models presents an inherent challenge to commercially available CCIPs that for all their advantages have one key limitation: there are none available on the market that have a biodegradable membrane. To overcome this limitation researchers at NIH have developed a technique that utilizes a biocompatible adhesive to attach an electrospun biodegradable poly(lactic-co-glycolic acid) (PLGA) membrane to the bottom of a cell culture insert. The production of these parts has become a standard practice to develop several tissue types, but it has limitations in that the process is laborious, time consuming and not scalable beyond a 24-well plate density. Topic Goals The goal of this project is to identify potential new biodegradable membranes that can be used to create CCIPs. Equally important is to identify manufacturing techniques that allow for the attachment of these custom membranes to CCIPs in a reproducible and cost-effective fashion without toxic adhesives or other contaminants in scalable well density formats (6, 12, 24, 96+). The optimal outcome would be a commercially available off-the-shelf 96 well CCIP that utilizes a biodegradable membrane. The availability of such a plate would increase the capabilities of groups conducting research that use CCIPs by potentially increasing the overall quantity, quality and viability of complex cellular constructs. Increasing the well density up to 96 wells would also push closer to true high throughput screening for groups using advanced cell models for in-vitro drug discovery. Phase I Activities and Expected Deliverables Phase I proposals must specify clear, appropriate, measurable goals (milestones) to be achieved. Phase I activities and deliverables may include the following: • Develop a prototype CCIP that has the following features: o Adheres as closely as possible to current ANSI/SLAS Microplate Standards  ANSI/SLAS 1-2004 (R2012) Microplates – Footprint Dimensions (formerly ANSI/SBS 1-2004)  ANSI/SLAS 2-2004 (R2012) Microplates – Height Dimensions (formerly ANSI/SBS 2-2004)  ANSI/SLAS 3-2004 (R2012) Microplates – Bottom Outside Flange Dimensions (formerly ANSI/SBS 3-2004)  ANSI/SLAS 4-2004 (R2012) Microplates – Well Positions (formerly ANSI/SBS 4-2004) o Utilizes membranes that have the following properties:  Biocompatibility with tissue culture environments  Biodegradability within a time period between 2-6 weeks  Suitable for cellular health and function for long term experiments (1+ month)  Have a thickness of <10 μm  Have a pore size <1 μm  Ability to increase cell attachment without the need for additional coatings o Incorporates a ridge or a cap on the underside of the insert such that it extends below the bottom of the membrane and ideally matches the inner diameter of the well wall. This in effect creates an additional well on the underside of the insert that provides greater structural integrity for three-dimensional tissues added to that portion of the insert.  This ridge or cap should extend no greater than 1mm from the bottom of the membrane layer. Page 68  This ridge or cap should match the inner diameter of the well wall as closely as possible to maximize the membrane surface area available for cell placement on the underside of the plate. o Utilizes manufacturing techniques and materials that do not introduce artifacts when undergoing standard high throughput screening measurements such as fluorescent microscopy. • Has at a minimum a 6 well density as a proof of concept. • Identify a tissue model to use as a standard to validate the functionality of the produced part. o This model should incorporate standard fluorescent labels such as DAPI, GFP, mCherry or others to determine if any of the materials used introduce a measurement artifact. o We have encountered certain adhesives that are biocompatible, although when introduced to a measurement system such as fluorescent microscopy the adhesive itself auto-fluoresces at the same emission wavelength as a cellular label such as GFP. This introduces a high degree of background signal that makes quantification of cellular features difficult if not impossible. This should be taken into consideration with regards to the identification of a tissue model to act as a means of validation. • Cost estimates to manufacture a device capable of meeting the specifications listed above. • Provide NCATS with all data resulting from Phase I Activities and Deliverables. Phase II Activities and Expected Deliverables • Build a prototype plate that meets the Phase I specifications with a 96 well density as a minimum. o This requires all of the necessary tooling and infrastructure necessary to manufacture the plate. • Provide a test plan to evaluate the Phase I validated tissue model in the 96 well density plate. o Provide NCATS with all data from each executed test to properly evaluate the model. • Develop a robust manufacturing plan for the plate, using off the shelf OEM components where possible to minimize expense. • Provide NCATS with all data resulting from Phase II Activities and Deliverables.
US Flag An Official Website of the United States Government