AVATAR workflow: Controlling cell destiny from start to finish
Simplify and improve your workflow with the primary cell type you’re working with. You’ll have consistently healthy viable cells at the end of each step, shorten your workflow timeline, and have more time to innovate instead. The AVATAR workflow is optimized for immune cells, stem cells, and tumor cells. Below is an example of the streamlined stem cell workflow from fibroblast to iPSC to motor neuron.
Step 1 – Transfect with high viability
Human primary dermal fibroblasts were transfected with reprogramming factors using AVATAR recovery settings. A 5X increase in transfection efficiency was achieved versus standard methods while also maintaining high cell viability.
Step 2 – Expand
Post-transfection with AVATAR also resulted in an increase in reprogramming efficiency. A 5X increase in iPSC colony formation was observed by day 19 using pressurized and hypoxic conditions.
AVATAR Setting #1
Step 3 – Maintain Stemness
AVATAR culture settings were next used to promote ‘stemness’ by inducing expression of Nanog, Oct4, Sox2. A combination of hypoxia and pressure (1% O2 and +2 PSI) maintained tightly-compacted colonies as compared to hypoxia alone and standard CO2 incubator. Expression of stemness markers Nanog, Oct4, and Sox2 increased 2-fold under hypoxic and pressurized conditions than standard or hypoxia alone (qPCR).
Expression of stemness markers Nanog, Oct4, and Sox2 increased 2-fold under hypoxic and pressurized conditions than standard or hypoxia alone (qPCR).
Step 4 – Targeted Differentiation
AVATAR culture settings were tuned to promote differentiation of iPSCs into Neural Progenitor Cells (NPCs) by inducing expression of early neuronal markers PAX6 and NESTIN.
Validating through targeted NPC gene expression, mRNA-seq revealed up-regulation of genes involved in neural differentiation and neural progenitor cell maintenance.
mRNA-seq of neo-natal iPSCs (left) cultured in AVATAR reveals up-regulation of genes involved in neural differentiation and neural progenitor cell maintenance and down regulation in pluripotency genes. Following qPCR further compliments mRNA-seq data for the indicated genes in the right panel.
Step 5 – Functional cells
Early expression of Synapsin 1 (SYN1) was seen by day 14 of neuron differentiation in the AVATAR, validating targeted protein expression for motor neuron maturation compared to those grown in standard conditions.