Development of Physiologically relevant Human Brain Models by Using Brain Decellularized Extracellular Matrix

Abstract

Introduction: In-vitro human brain models have proven to be effective for platforms for drug screening as they reconstruct pathophysiological features found in brain diseases. Conventional culture systems, however, rely on synthetic or natural bioinks derived from tissues, other than brains, and therefore, they lack most of necessary components in the native microenvironment. In this regard, decellularized extracellular matrices have recently emerged as promising bioinks to combat this limitation. To facilitate more accurate pathophysiological study and drug screening, a recently developed our porcine derived brain decellularized extracellular matrix (BdECM) was combined with Matrigel, one of the gold standard hydrogels, and employed to our validated microfluidic human brain model in order to recapitulate microenvironment found in human brains.

Materials and Methods: The BdECM was synthesized through decelluarization of porcine brains. The mixture of BdECM and Matrigel hybrid hydrogel was incorporated into ourthe recently developed our microfluidic models consisting of neurons, astrocytes and the microglia. To validate the model, we assessed microglial immune response by performing immunocytochemistry for CD11B and CD206. Furthermore, Tto quantitatively measure potential cytotoxicity of BdECM, lactate dehydrogenase (LDH) assay was performed.

Results and Discussion : The gelation kinetics of BdECM hydrogel was tested at concentrations of 0.5, 1, 2, and 4% of BdECM. The results showed that BdECM was capable of achieving a storage modulus from 100 to 200Pa, which is comparable to the modulus found in human brain tissues. This indicates the inclusion of BdECM in the cell culture hydrogel may further improve the physical properties of conventional hydrogel, such as Matrigel. To check the physiological relevance of the combination of Matrigel and BdECM, we assessed the cytotoxicity of the mixture and found that the cytotoxicity of Matrigel and the mixture was 1.9 and 2.3% respectively indicating there was no significant increase in cytotoxicity with the addition ofthere was no significant cell death compared to Matrigel, as seen in Figure 1 BdECM. In addition,Even though the permeability of BdECM+Matrigel (0.0104 ± 0.0051 cm/s) was comparable tolower that of Matrigel (0.013 cm/s and 0.00678 ± 0.01395 cm/s respectively), the limited nutrient transport through the gel did not affect the cell viability. In addition, no immune response was found when the microglia cultured on BdECM, determined by microglial activation markers, such as CD11B and CD206.

Conclusions: Our results showed that BdECM hydrogel will provide relevant stiffness to native brain and the hybrid of BdECM and Matrigel provided microenvironments for brain cells without causing significant cell death and immune response. We are expecting that this gel recipe will offer a promising cell culture platform for in vitro brain models, which closely recapitulate human brain.