(B-55) Cell-derived matrix hydrogels allow for generation of human vessel-on-chip system

Introduction:: Decellularized extracellular matrix (dECM) scaffolds and hydrogels are commonly used in in vitro and in vivo tissue engineering techniques to more faithfully recapitulate the native microenvironment.¹ dECM is typically harvested from animal tissue and decellularized using a variety of different methods, but the use of human dECMs has been limited due to the availability of human tissue.² Cell-derived matrix (CDM) can be generated from many different human cell types in vitro and provides a platform to study the biophysical and biochemical properties of ECM.^2,3 In this study, we develop a dECM hydrogel platform synthesized from CDM from human dermal fibroblasts. We then demonstrate the utility of this hydrogel within a microfluidic device to fabricate a vessel-on-chip platform that incorporates complex human derived ECM and human endothelial cells for the first time.


Materials and Methods:: CDM was first generated from commercially available human dermal fibroblasts by treating cells plated at confluence with 50 µg/mL ascorbic acid daily for 6 days. After 7 days, CDM was decellularized, powdered with an LN₂ cooled mortar and pestle, and lyophilized to remove excess water. CDM powder was then solubilized in 0.6 mg/mL pepsin in 0.01M HCl to generate a pre-gel solution. CDM+COL-I hydrogels were then formed by combining CDM pre-gel with collagen I (COL-I) in a 4:1 ratio and adjusting the isotonic balance and pH of the solution. As a control, COL-I hydrogels were formed from rat tail COL-I in the same manner. All hydrogels were made with a final protein concentration of 7.69 mg/mL. The composition of the resulting hydrogels was analyzed using proteomics, the gelation kinetics were measured by light scattering, the mechanical properties were measured by nanoindentation, and the nano- and microstructure was analyzed by scanning electron microscopy (SEM). To incorporate CDM hydrogels into a vessel-on-chip platform, 160 µm diameter acupuncture needles were dip coated with 2% gelatin and then inserted into devices, after which CDM+COL-I or COL-I hydrogels were injected into the device and then incubated at 37C. Needles were removed after 24 hours, and the vacant channel left behind was washed with media prior to seeding with either human umbilical vein endothelial cells (HUVEC) or blood originated endothelial cells (BOEC) suspended in media at a concentration of 2×10⁶ cells/mL. 24 hours after seeding, vessel-on-chips were fixed and stained for F-actin and DNA.


Results, Conclusions, and Discussions:: We confirmed gelation time for CDM+COL-I hydrogels to be similar to that of COL-I hydrogels alone, while CDM hydrogels without the addition of collagen had a longer gelation time that did not result in a mechanically stable hydrogel (Fig 1a). Young’s modulus of COL-I hydrogels was significantly higher than that of CDM+COL-I hydrogels (Fig 1b). Proteomics revealed that while there was some loss of proteins from lyophilized proteins to pre-gel solution, many structural proteins including native human collagens were maintained in the CDM hydrogels. Both CDM+COL-I and COL-I hydrogels were capable of gelling within a microfluidic device and maintained a cylindrical vacancy within the gel. This vacancy was able to be seeded with HUVECs and BOECs and maintained for 24 hours post seeding (Fig 1c, d). In this work, we have utilized human cell-derived matrix to generate a hydrogel that retains protein complexity and can be used as a cellular substrate in a similar manner to traditional COL-I hydrogels. We have characterized the mechanical properties of the CDM hydrogel and used the resulting platform to generate a vessel-on-chip platform from human derived components.




Acknowledgements (Optional): : This work has been supported by NIH R35GM142944 (WJP), NIH T32HL69768 (ELD), and NIH F31HL162462 (ELD).


References (Optional): : 1. Zhang W. Regenerative Therapy. 2021 Dec 1;18:88-96.
2. Assunção M. Frontiers in bioengineering and biotechnology. 2020 Dec 3;8:602009.
3. Tello M. Methods. 2016 Feb 1;94:85-100.