Cytocaf Technologies

What is Caf1?

Chromatin Assembly Factor Complex 1 (Caf1) is a protein which is produced by bacteria, forming a gel-like coat that protects them from phagocytosis.

FIBRONECTIN (Caf1 - Fibronectin)

Caf1-Fibronectin mimics the action of the extracellular matrix protein fibronectin, providing an adhesive surface for cells to stick to. Caf1-Fibronectin supports the growth of multiple cell types, including mesenchymal stromal cells (MSCs) and human dermal fibroblasts. Fibronectin-Caf1 benefits from the modular nature of the Caf1 polymer, combining easily with other types of Caf1 subunit to create multifunctional polymers or tune the level of cell adhesion.

Caf1-Fibronectin hydrogels support the growth of human dermal fibroblasts. Hydrogels of non-adhesive Caf1 wild type (Caf1- WT) and adhesive Caf1- fibronectin were constructed in wells of a 24-well plate. Cells were seeded on top of the hydrogels and grown for 7 days before staining and visualization using a confocal microscope (green - live cells, red - dead cells). Right: Caf1-Fibronectin coated surfaces support the growth of human MSCs. Wells of a 24-well plate were coated Caf1-Fibronectin or recombinant fibronectin or left uncoated. Wells were then seeded with human MSCs and grown for 7 days, with cell counts measured using the PrestoBlue assay.

Mosaic Caf1 Polymers

Mosaic Caf1 polymers that contain more than one type of subunit can be easily made through thermal refolding of the polymers. Here, non-adhesive Caf1 wild type (WT) was mixed in different ratios with adhesive Caf1-Fibronectin, and used to coat surfaces of a 96 well plate. Cells from different cell lines were then seeded onto the surfaces and grown for four hours before fixation, staining and counting. The number of adherent cells was proportional to the ratio of Caf1-Fibronectin subunits in the polymer.

VITRONECTIN (Caf1 - Vitronectin)

Caf1-Vitronectin mimics the action of the extracellular matrix protein vitronectin, supporting the adhesion, growth and maintenance of cell types such as induced pluripotent stem cells (iPSCs). iPSCs grown on Vitronectin-Caf1 form colonies with morphologies similar to those seen on recombinant vitronectin and Geltrex™, with no significant differences in cycle length or population doublings. Furthermore, they retain their stemness markers and capacity to differentiate into different cell types.

Analysis of pluripotency markers of hiPSCs grown on Caf1-VTN compared to Geltrex. Cells were expanded over repeat subcultures then probed for transcription factors OCT4 (purple), NANOG (green), and surface antigen TRA1-60 (red) to highlight endogenous expression of pluripotency marker genes. Scale bar: 50µm.

Embryoid body formation from iPSCs grown on Caf1 substrates and Geltrex

Embryoid body formation from iPSCs grown on Caf1 substrates and Geltrex show differentiation into the three germ layers. Heat map of gene expression fold changes from TaqMan hiPSC scorecard assay with mesoderm, ectoderm, and endoderm gene markers highlighted. Box plots show centre quartiles for all genes in a lineage set. Significance determined by one way ANOVA with Šídák's correction, **** p < 0.0001.

Reference: Engineering bacterial protein polymers to support human pluripotent stem cell growth and differentiation in culture. Creigh AR et. Al., bioRxiv 2024.04.29.59160