Poster BB19 - Development of a Scalable, Microcarrier-based Approach to Produce Human Cell-Derived Matrix Hydrogels

Introduction: Human cell-derived matrix (hCDM) hydrogels provide an approach to recapitulate the native protein and structural complexity of the in vivo microenvironment. Patient-derived, hCDM hydrogels offer an individualized and clinically relevant platform for in vitro disease models. Our lab has developed a protocol to generate compositionally complex and bioactive hCDM hydrogels using human dermal fibroblasts. However, this protocol is lengthy, labor intensive, and has low yield. In this study, we adapt the existing hCDM protocol to a 3D, microcarrier-based, suspension culture to increase hCDM output by increasing cultured surface area. This approach aims to improve the clinical translatability of in vitro disease models through the development of a scalable approach for generating patient-specific extracellular matrix.

Materials and

Methods: Cytodex 3 (120-170 µm diameter) microcarriers (MCs), composed of a dextran matrix core with a denatured collagen coating, were prepared according to manufacturer’s instructions at 5 mg/mL in culture media. Human dermal fibroblasts were mixed with the microcarrier solution in FACS tubes to achieve a final cell concentration of 2x105/mL resulting in a cell:MC ratio of 10:1. Cells were seeded to MCs for 4 hours at 37º C, mixing every fifteen minutes, then transferred to a spinner vessel (60 RPM) with a total volume of 25 mL. For seeding optimization, the seeding procedure was performed again after culturing for 24 hours. 50 ug/mL ascorbic acid and media replacements every other day. MCs were decellularized after seven days using triton-X-100 and ammonium hydroxide followed by DNase. To evaluate decellularization, samples were stained with DAPI and Rhodamine Phalloidin.

Results, Conclusions, and Discussions: The MC suspension approach provides an increase in cell culture surface area. A 25 mL suspension culture has roughly 337 cm2 of culture surface area and can be scaled to 100 mL culture by increasing spinner vessel volume. Our lab’s previous protocol generates hCDM in batches of 10 x 100 mm dishes with 567 cm2 surface area, requiring a total of 400 mL of media for the 7-day culture. One 100 mL spinner vessel would have 1350 cm2 of culture surface area and require 440 mL of culture media for an equivalent 7-day culture. A 25 mL suspension culture with a double inoculation requires 10 million cells (3.0x104 cells/cm2) compared to the established method which requires 2 million cells per 100 mm dish (3.5x104 cells/cm2). The double inoculation increases the number of cells attached to MCs from Day 1 to Day 2, shown through DNA/F-Actin immunofluorescent imaging (Figure 1A). However, a large portion of seeded cells did not attach, suggesting cell inoculation efficiency can be further improved. MCs on Day 7 mainly merged into clumps of 2-10 MCs forming cell networks around and between MCs with notable filamentous actin structures (Figure 1B). After Decellularization, MCs clumps of >20 MCs were common. Decellularization removed nearly all F-Actin, shown through a significant reduction in mean phalloidin fluorescence (Figure 1C). Patterns in DAPI stained images clearly identify cell nuclei throughout the MCs on Day 7. After Decellularization, the circular nuclei structures become infrequent as MCs more-commonly had fibrous-like streaks of positive DNA staining on and between MCs. This potentially indicates an ineffective DNase washing step during the decellularization protocol.
Ongoing efforts are focused on the quantification of hCDM and optimization of the harvesting. Further optimization of cell inoculation, decellularization and hCDM harvesting should improve the overall yield efficiency to meet the established protocols hCDM yield of 10 µg/cm2 culture surface area. Improvements to the per cell yield will be prioritized for patient-specific hCDM, where cells are limited. After validating the new MC-based protocol further hCDM development can be pursued, including the investigation of other patient cell types compatible with this approach.

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