Lifeng Kang
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High Throughput Study of Cell-ECM Interactions in 3D Environment Using Microwell Arrays
The extracellular matrix (ECM) is critical in developing an integrated picture of the role of the microenvironment in the fate of many cells. A two-dimensional (2D) microarray method was reported for cell-ECM interaction study [1]. These two-dimensional approaches can be complemented by three-dimensional (3D) approaches such as embedding cells within ECM gels [2]. However, 3D microarray methods are difficult to develop due to difficulties such as ECM array fabrication and nanolitre liquid handling. To overcome these difficulties, microwell array and robotic spotting may be useful.
In this study, we develop an approach using a microarrayer (Piezorray) and microwell arrays for cell-ECM interaction study with high throughput. The microwell array was fabricated with soft lithography. The diameter of microwell was 400 µm with a pitch of 600 µm. In total, 2100 microwells were fabricated on a single slide with numbers and alphabets in between for identification. As a proof-of-concept experiment, it was shown that dye solutions can be printed accurately into these microwells proloadd with collagen solution. For future study, we will print ECM component in a combinatorial manner into the microwell array preloaded with cells in prepolymer solutions. Then the mixtures will be UV-crosslinked to immobilize the ECM mixture inside each isolated microwells for cell-ECM interaction study.
i0000000000@gmail.com
Research Interests:
Biomaterials
Experience & Skills:
Drug delivery and tissue engineering
Meeting Summary (Help)
Meeting Date: Jan 6, 2008
Summary: The figures: 1) schematic, 2) Homogeneity of the mixture, 3) Stability of the microogels, and 4) Biocompatibility of the microgels
Action Plan: To continue stability studies for fibronectin and laminin in HA microgels and to quantify the data by measuring the fluorescent intensity
Progress on issues from last meeting:
Showed that collagen microgels were stable in PBS for 3 days.
Ali's comments:
Transfer the information
Meeting Date: Dec 11, 2008
Summary: To make HA and collagen mix, mechanical forces are required.
So the ECM components are to be mixed with HA before loading to the microarrayer.
However, the increased printing workload made printing difficult.
The mixture considered good was dismissed since there are slumps inside.
Action Plan::Much work needs to do on how to mix and check the native morphology of collagen in tissue.
To test the consistency of the array printing into microwells of the HA-collagen mixture.
Progress on issues from last meeting:
Meeting Date: October 28 2008
Summary: Continuing microarray work
Action Plan:
Meeting Date - October 19 2008
Summary
Schedule regular micro-array meetings.
High cell viability after 2 hours. Are there more problems to solve ? Is there a plan ? Can someone take charge organizing this ?
Bring figures to discuss.
Organization of tasks. Leader for the team ? Plan for transition ?
Collagen printing as a 'knot'. Plan for diffusion ? Concentration of acetic acid ?
Meeting Date : Sept 26 2008
Summary:
(1) The staining of collagen I, collagen IV and chondroitin in microwells. No spots could be detected by the microarray scanner.
Possible solutions: validate the primary antibodies and the staining process.
(2) Whether HA gels can immobolize ECM components
Possible solutions: confocal images can be taken after staining to show the existence of the ECM components in gels.
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