axoCells™ human iPSC-derived microglia (unaffected), male donor, aged 40-50, ≥1 million cells

Phenotypic characterization: Morphology

axoCells iPSC-derived microglia demonstrate appropriate morphology, with variable phenotypes demonstrating different populations of microglial subtype.

Cryopreserved axoCells microglia (ax0664) were thawed and matured for 7 days on Surebond XF + concanavalin A.

Phenotypic characterization: ICC

axoCells microglia have been extensively characterized for expression the key markers Iba1, TMEM119, CX3CR1 and P2RY12.

axoCells iPSC-derived microglia were thawed and matured for 7 days. Cells were fixed and stained for the relevant markers and imaged using a Leica microscope. Key microglia markers Iba1, TMEM119, CX3CR1 and P2RY12 were used as standard quality control. Red: marker of interest, Blue: DAPI (neuronal marker)

Phenotypic characterization: Flow Cytometry

As part of our rigorous QC, we include flow cytometry as standard to demonstrate committal down the myeloid lineage, with SSEA4 (a marker of immaturity) as a negative control.

Flow cytometry QC for fresh macrophage progenitors (monocytes). Progenitor cells from all batches undergo QC for a standard panel of markers.

Functional relevance: Cytokine release

As part of our suite of functional assays, we have extensively characterized our cytokine release assay to demonstrate the functional relevance of axoCells microglia.
A) This graph demonstrates increasing response to LPS with expected relationship. B) Functional relevance demonstrated by cytokine release in response to variable stimulation.

A) IL6 release in fresh iPSC-derived microglia following 24 h stimulation with LPS concentration curve (n=3). B) Cytokine release from iPSC-derived microglia following 24 h stimulation with LPS, INFy or both (n=3). UNS= unstimulated.

Functional relevance: High-throughput cytokine release assay

As part of our functional characterization, we are able to conduct high-throughput cytokine release assays with a comprehensive cytokine panel using O-link Technology.

This data represents expected cytokine release in response to pro-inflammatory (LPS, INF-gamma) and anti-inflammatory (IL4) stimulation.

Cytokine release following microglia stimulation. Fresh microglia were matured for 7 days prior to stimulation by LPS, INFy or IL4 for 24h. Cell supernatant was collected and analysed by O-link technology. Table shows summary of how cytokine release was affected by each stimuli.

Functional relevance: Phagocytosis

We have extensively characterized our phagocytosis assay across a range of baits, meaning we are able to select appropriate baits based on experimental need. This includes disease-associated baits such as alpha-synuclein (important for Parkinson’s Disease modelling) and beta amyloid (relevant for Alzheimer’s Disease).

Other bait such as E. coli, zymosan beads, TAU and myelin basic protein, can be labelled and tested on request.

Phagocytosis of various pHrodo labelled bait by fresh iPSC derived microglia. Cells are matured for 7 days and phagocytosis performed by adding bait to the cells and monitoring update over time using an IncuCyte S3. S. aureus (ThermoFisher), iPSC-derived dead neurons (generated and labelled in house) and alpha-synuclein (labelled in house).

axoCells microglia were thawed and matured for 7 days before addition of pHrodo labelled bait. Myelin basic protein, beta-amyloid and S. aureus were added to the cells and phagocytosis monitored over 24h using an IncuCyte S3, showing a steady increase over time. Cytochalasin D (10uM) was used as a negative control, and showed complete inhibition of phagocytosis.

Functional relevance: Chemotaxis

As part of our suite of functional assays, we have extensively characterized our chemotaxis assay for use in axoServices projects and compound screening. axoCells microglia demonstrate expected chemotactic response to physiological agents, demonstrating their functional relevance.

Chemotaxis by fresh iPSC-derived microglia to various concentrations of C5a. iPSC derived microglia were matured for 7 days before re-plating into chemotaxis plates. A range of concentrations of C5a were used as a chemoattractant and cell movement was measured using an IncuCyte S3. Values represent number of cells moving from the top chamber to the bottom, towards C5a, after 24h. Data are n=4 +/- SEM.

Comparison of healthy control and ALS-derived microglia

Microglia derived from axoLines disease iPSCs can be tested against healthy control cells in assay format, with measurable endpoints essential for effective disease modelling and compound screening. In this way, they can be incorporated into human-relevant models of neuroinflammation and neurodegeneration, such as Alzheimer’s Disease, Parkinson’s Disease and ALS.

Fresh iPSC derived microglia from healthy or ALS (C9orf72) background were matured for 7 days and phagocytosis of myelin basic protein (MBP) assessed. MBP was labelled with pHrodo dye and added to the cells. Phagocytosis was quantified using an IncuCyte S3 for up to 48h (A). B) shows data after 24h from addition of bait. A t-test was perfomed to assess any statistical significance between the cell lines * p<0.05.

Applications

Our axoCells^TM microglia have been specifically developed for use in in vitro disease modeling and drug discovery. As inflammation is linked to many neurological diseases such as Alzheimer’s, Parkinson’s, ALS, and aging, microglia are becoming targets for treatments of these conditions.  

Axol’s production method mimics the in vivo pathway of development for brain resident macrophages and produces cells that are functionally representative of primary human microglia in vitro. Our microglia have been used extensively in co- and tri-culture with Axol’s cortical excitatory neurons, astrocytes, and/or inhibitory interneurons to produce more relevant models of human diseases such as Alzheimer’s and Parkinson’s. 

Protocols

 Protocols for incorporating microglia into co- and tri-culture with neurons are available as well as for microglia in monoculture. 

Quality

We maintain or exceed industry-level quality with our ISO:9001-accredited production facility, guided by our rigorous quality control procedures and decades of scientific experience.  

All cells come with a full Certificate of Analysis and are officially certified by HPSCreg®, to ensure ethical and biological conformity for your peace of mind. 

Licenses & Consent

Axol has obtained all relevant licenses for reprogramming donor samples into iPSCs and differentiating these into microglia for commercial use.  

Patient samples used to create these cells have been ethically sourced and consented for research and commercial use. All cells come with a full Certificate of Analysis and are officially certified by HPSCreg®, to ensure ethical and biological conformity for your peace of mind. 

Suzanne Edavettal, et al  2022. Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis, Med, Volume 3, Issue 12, 2022, Pages 860-882.e15, ISSN 2666-6340, https://doi.org/10.1016/j.medj.2022.09.007.

Timothy J. Sargeant, Célia Fourrier 2023. Human monocyte-derived microglia-like cell models: A review of the benefits, limitations and recommendations, Brain, Behavior, and Immunity, Volume 107, 2023, Pages 98-109, ISSN 0889-1591, https://doi.org/10.1016/j.bbi.2022.09.015.

McKee Chloe G., Hoffos Madison, Vecchiarelli Haley A., Tremblay Marie-Ève 2023. Microglia: A pharmacological target for the treatment of age-related cognitive decline and Alzheimer’s disease. Frontiers in Pharmacology 14 2023. http://doi.org10.3389/fphar.2023.1125982

Media & Reagents

A ready to go kit containing microglia cells and optimized media is available Microglia Complete Cells and Media (ax0679) or media is available as a standalone item Microglia maintenance media kit (ax0660).