Brain organoid formation on decellularized porcine brain ECM hydrogels (1)
Most commonly, Matrigel is used as an outer hydrogel scaffold to give brain organoids structural support and to supply the organoid tissue with differentiation factors. However, the composition of Matrigel differs from the composition of the extracellular matrix (ECM) of the native brain and is therefore not specific. The ECM of the brain is unique in its composition, as it contains less fibrous structural proteins, such as collagen or elastin, and more proteoglycans compared to other tissues. Lecticans, which are a subfamily of proteoglycans, are especially abundant in the brain ECM. Decellularization (DC) is the process of removing cells from the ECM by chemical, physical, and enzymatical methods.
Native pig brains were obtained from a commercial supplier, delivered frozen in plastic cups, hence the cylindrical form. Histological staining with DAPI showed only a few or no nuclei in the DC samples. Some remnant DNA molecules are still present in the ECM. The content of sulfated GAGs was significantly decreased in the DC sample compared to the native sample. the soluble collagen content was significantly increased in the decellularized sample compared to the native sample. The DC process removed more than 90% of the nuclear material and structural proteins such as collagen were retained. However, functional proteins such as GAGs were partly washed out during DC.
Mass spectrometry analysis showed that decellularization and hydrogel processing removed most proteins except for collagen, which made up more than 90% of total protein abundance in both the B-ECM and the B-ECM hydrogel group. In the native ECM, 101 proteins included in the matrisome (ECM proteins and ECM-associated proteins) were detected, of which 64 are ECM-associated proteins, and 37 are ECM proteins consisted mostly of glycoproteins and proteoglycans. In B-ECM, 91 matrisome-proteins were detected, of which 60 are ECM proteins, including 16 collagens, 31 glycoproteins, and 13 proteoglycans. The collagens account for about 90% of the matrisome-proteins abundance in B-ECM, while they account for less than 5% in the native ECM. In B-ECM hydrogel, 18 matrisome-proteins were detected, of which 15 were ECM proteins including six collagens (COL1A1, COL1A2, COL2A1, COL3A1, COL5A2, COL6A1), that accounted for greater than 95% of the abundance of the matrisome-proteins, eight glycoproteins (FBN1, FBN2, FGB, LAMB1, LAMB2, LAMC1, SRPX, and VWF), and one proteoglycan (HSPG2). All ECM proteins except FBN2 and VWF overlapped between the B-ECM hydrogel and the B-ECM group.
Correct neuroectodermal development in the B-ECM hydrogel brain organoids was observed. NESTIN, TUBB3, DCX, MAP2 and GFAP as relevant neural markers showed no significant differences in gene expression, indicating equal effect of Matrigel or B-ECM on gene expression. Radial glia cells (stained with SOX2+, Nestin+, PAX6+) were located in the ventricular like zone of the organoids, from where they have been found to start migrating towards the outside while maturing into neurons (stained with β-tubulin III+, DCX+, MAP2+).
Different DC protocols were tested based on combinations of SDS, TritonX or sodium deoxycholate (SDC) as well as DNA removing enzymes. A new protocol based on the ionic detergent SDC and DNase was established. SDC proved to be a suitable DC detergent for the large brain volume, with little or no cellular remnants or antigens detectable after DC. The use of SDC-based protocols has previously shown to be beneficial over SDS-based protocols, as increased damage to ECM proteins in different tissue DC applications has been reported with SDS. The B-ECM and B-ECM hydrogel group had a high abundance (greater than 90%) of collagens. Collagen is less abundant in brain tissue compared to other tissue types, however different collagen types are present in parts of the brain such as the microvasculature. Loss of GAGs in the DC process may in part be explained by the removal of cells, which contain membrane–bound GAGs. Removal of GAGs is seen as unbeneficial, as GAGs can guide cell behavior. However, GAG removal is a common result of detergent/enzyme based DC, and has been observed in multiple different studies.