ioCells are human cells deterministically programmed from induced pluripotent stem cells (iPSCs) using bit.bio’s patented opti-ox technology. opti-ox enables the programming of entire stem cell cultures into a precise cell identity with unrivaled lot-to-lot consistency; this, coupled with bit.bio's industrial manufacturing capabilities that facilitate production at scale, ensures reproducible results across experiments.
bit.bio ioCells are derived from human iPSCs using opti-ox technology (as described in Pawlowski et al. 2017), which relies on the precise genetic engineering of iPSCs with transcription factors (TF). Induction of TF expression leads to the irreversible loss of pluripotency and the synchronised homogeneous production of human iPSC-derived cells that are primed and committed to a specific cell fate.,
The parental human iPSC lines used to generate ioCells originate from human donor cells such as fibroblasts or endothelial progenitor cells, which have been reprogrammed using the four retrovirally transduced Yamanaka factors (OCT4, SOX2, KLF4, MYC).
The human iPSC parental lines used by bit.bio have been generated from specimens that were collected with the informed consent of the collection site and the individual donors. For further details, please refer to The Limited Use License & Statement of Use. For specific donor details, such as gender, race, and age, please refer to the specific product of interest.
If the product of interest is not available in the catalogue, bit.bio also offers services for the custom development of iPSC-derived cells and derivative models, such as disease model cells including a disease-relevant mutation. These iPSC-derived disease model cells can be paired with their corresponding genetically-matched control to offer a physiologically relevant model to investigate the effect of the mutation of interest. For custom requests and services, please contact us.
ioCells are delivered in a cryopreserved format and typically shipped on dry ice (liquid nitrogen shipments can be arranged upon request). Immediately upon arrival and until use, the cells should be stored in liquid nitrogen vapour phase or ultra-low temperature freezers (-150°C). The cells are ready for experimentation within days of revival in the recommended media. For information about the number of cells per vial and pack sizes, refer to the specific product of interest.
ioCells are cryopreserved in a medium containing 10% DMSO to support viability upon thaw. For specific compositions please refer to the product specific Safety Data Sheet (SDS).
ioCells must be stored in liquid nitrogen vapour phase or ultra-low temperature freezers (-150°C) immediately upon arrival and until use. bit.bio guarantees the quality of the product for 12 months from delivery when stored and used correctly.
Lot-to-lot consistency data for ioCells is generated by bit.bio using bulk-RNA sequencing. The transcriptomic analysis of the bulk RNA-sequencing data reveals tight clustering of lots, demonstrating high consistency across time points. Data for each wild-type cell can be found on its product page under Technical data.
Every lot of ioCells produced by bit.bio adheres to a rigorous quality control (QC) process. The standard QC procedures include sterility testing, morphological assessments, immunocytochemistry staining (ICC), and either RT-qPCR or a functional assay (such as phagocytosis) to ensure quality and consistency. A lot-specific Certificate of Analysis (CoA) with details of the QC carried out is provided with every product.
bit.bio follows strict aseptic bio-banking procedures during the manufacturing of ioCells. As part of quality control (QC), each manufactured lot is tested for sterility (microbial and fungal) and for the absence of mycoplasma (pan species) using industry-standard validated methods. For specific details per manufactured lot and product, refer to the Certificate of Analysis (CoA). Viral clearance documentation for each product is available on request.
Certificate of Analysis (CoA) and biosafety documentation are available for all lots of bit.bio ioCells. Contact us to request the specific documentation of interest.
The genotype and mutation status for each ioDisease Model Cells product can be found on the specific product page under the section product specifications.
bit.bio recommends using the reagents listed in the cell type-specific user manuals as they have been routinely used and validated with ioCells. While reagents from other suppliers with identical specifications should lead to similar results, bit.bio’s quality control (QC) and cell profiling are performed using the recommended reagents. We cannot guarantee alternative reagents will perform to the same specifications as those tested by bit.bio.
bit.bio recommends using the reagents listed in the cell type-specific user manuals for optimal results as these have been routinely used and validated with ioCells. The use of animal-free reagents may require optimisation of cell culture conditions.
ioCells culture media are open-source to provide scientists with transparent information about cell culture conditions. Each cell type-specific user manual provides the list of cell culture reagents, including supplier and catalogue number, which have been validated and are routinely used at bit.bio.
Cell culture media kits for ioGlutamatergic Neurons and ioMicroglia are available for purchase from bit.bio. For more information, refer to the product pages.
Reagents should be stored according to the recommendations provided in the cell type-specific user manual. In general, reagents should be aliquotted into single-use volumes and stored at the recommended temperature to avoid freeze-thaw cycles. Do not continue using the reagent past its expiry date. For additional enquiries about specific reagents, refer to the manufacturer’s instructions.
Storage recommendations for reagents and growth factors can be found in the cell type-specific user manual or in the specific assay protocol. Do not continue using the reagent beyond its expiry date. For additional enquiries, refer to the manufacturer’s instructions.
Stock solutions should be prepared according to the cell type-specific user manual or the specific assay protocol. Stock solutions should be aliquoted into appropriate volumes of single-use aliquots to prevent freeze-thaw cycles.
bit.bio recommends culturing human iPSC-derived cells on tissue culture-treated plates. At bit.bio, ioCells are routinely cultured on the following plates:
Specific applications may require different culture plates; in these cases, bit.bio recommends optimising coatings and seeding densities to ensure successful cell culture.
bit.bio cell type-specific user manuals and protocols are optimised for culture of ioCells on tissue culture-treated polymer-bottomed plates.
For immunocytochemistry (ICC) and live-cell imaging applications, bit.bio routinely cultures ioCells on high-quality polymer-bottomed imaging plates such as ibidi µ-plates #1.5 polymer ibiTreat and Revvity PhenoPlate™.
bit.bio imaging protocols are not optimised for culturing on glass, so this approach is not currently recommended and would be at the user’s own risk.
bit.bio does not recommend the use of pre-made coatings or pre-coated plates for the culture of ioCells. Refer to the cell type-specific user manual for recommended plate coating reagents and step-by-step coating procedures.
bit.bio recommends following the cell type-specific user manual for step-by-step guidance on thawing ioCells. In general, remove the cryovial from liquid nitrogen storage immediately before thawing and keep it on dry ice for a minimal amount of time before moving it into a water bath warmed to 37°C. Keep the vial immersed without submerging the cap and maintain a constant gentle agitation.
The cryovial is ready for the next step when only a very small fraction of ice is left visible (recommended thaw times can be found in the user manual).
To ensure optimal cell recovery, use a 37°C water bath to thaw cells. Avoid bead baths as they do not provide the uniform heating required for proper thawing.
bit.bio recommends thawing a maximum of 3 vials of ioCells at once per handler to ensure rapid and consistent processing. These vials can be pooled into the same conical tube for the subsequent centrifugation and counting steps. See the cell type-specific user manual for more details.
When thawing bit.bio ioCells, the recommended basal medium should be warmed to room temperature to avoid shocking the cells during the cryovial wash and centrifugation steps. In addition, the medium that the cells will be seeded into should be pre-warmed to 37°C. Refer to the cell type-specific user manual for full guidance.
To ensure accurate cell counts, bit.bio recommends resuspending the cell suspension thoroughly by gently pipetting up and down; this avoids clumping and ensures a uniform single-cell distribution. Perform three separate counts of the cell suspension to improve reliability and detect any inconsistencies. For practical guidance on cell counting, please refer to the cell counting protocol and workshop.
ioHepatocytes should be treated gently with minimal pipetting. Follow the ioHepatocyte-specific user manual for guidance.
bit.bio recommends counting cells with an automated cell counter and including a viability dye such as Trypan Blue or AO/PI to ensure an accurate count and plating of viable cells. bit.bio recommends using AO/PI when possible, as it ensures that debris and non-nucleated cells do not interfere with cell counts. For recommended seeding densities, always consider viable cells. For further guidance on how to count ioCells, refer to the cell counting protocol.
Cell viability is highly dependent on counting methodology and thawing technique. If you have strictly followed the cell type-specific user manual and are still experiencing inconsistent viability across vials, record the viability and lot number and contact our technical support team.
bit.bio recommends following the cell type-specific user manual for seeding density. It is important to note that the recommendations in the user manuals are for monoculture. bit.bio recommends optimising seeding densities for specific assays, applications, or multi-cultures. Visit our protocol library for in-house validated applications.
For guidance on cell health and morphology at various time points, refer to the videos and images available on the product pages and to the cell type-specific user manuals.
bit.bio recommends following the cell type-specific user manual for guidance on medium changes, as the frequency varies depending on the cell type. Using fresh pre-warmed medium at the suggested intervals supports optimal cell health and performance.
For optimal results, media should be freshly prepared and pre-warmed to 37°C on each day of feeding. bit.bio provides guidance on media preparation and storage in the cell type-specific user manuals. Reagents and growth factors may be prone to degradation or sensitive to light, requiring careful storage to maintain activity. For further details, refer to the manufacturers’ instructions.
bit.bio does not recommend deviating from the feeding schedules provided in the cell type-specific user manuals. Maintaining the suggested intervals and using freshly supplemented medium is essential to guarantee optimal culture health and experimental consistency. For some cell types, we have suggestions for weekend-free schedules, please refer to the cell type-specific user manual and protocols for complete details.
While the addition of penicillin-streptomycin is generally compatible with human iPSC-derived cells, bit.bio has not formally validated their use with our products. Note that antibiotics can interfere with specific applications, such as MEA recordings, or mask underlying low-level contamination. To ensure culture integrity, we recommend relying on stringent aseptic technique within a laminar flow hood rather than a continuous use of antibiotics.
Doxycycline is a necessary reagent for ioCells stabilisation and, for some cell types, it is required for the extended cell culture. bit.bio recommends following the guidance in the cell type-specific user manuals and using the listed reagents at the recommended time points.
With the exception of ioOPC-like cells, bit.bio ioCells are designed for single use only, and passaging the cells may compromise their identity, viability, and/or functionality. ioOPC-like cells remain proliferative for a 2-week period and can be passaged following the exact user manual recommendations.
bit.bio ioCells are cryopreserved in a state primed for immediate programming to the specific cell identity. With the exception of ioOPC-like cells, they are provided experiment-ready for single use. bit.bio does not recommend further freeze-thaw cycles for any ioCells products, as this may compromise cell identity, viability, and functionality.
bit.bio validates ioCells using RT-qPCR, bulk RNA-sequencing (RNA-seq) and single cell RNA-seq. Our bulk RNA-seq data demonstrates high consistency across multiple lots and time points. For information on the expression of genes of interest, contact our team with the cell type and gene name(s). To support scientists in their initial experimental design, bit.bio provides data for up to 50 queries across our cell types free of charge.
bit.bio routinely performs RT-qPCR as quality control (QC) for most ioCells. For a step-by-step guide on RNA extraction and RT-qPCR, refer to the bit.bio RT-qPCR protocol. bit.bio does not currently have an established protocol for RT-qPCR for ioMicroglia.
bit.bio ioCells are fully compatible with standard immunofluorescence (IF) and immunocytochemistry (ICC) techniques. To help you achieve high-quality results, cell type-specific protocols for immunostaining and fluorescence imaging are available in our protocol library.
bit.bio routinely performs immunocytochemistry (ICC) staining during quality control (QC). Protocols for immunostaining and fluorescence imaging that include details of validated antibodies are available in the protocol library.
bit.bio ioCells are suitable for multi-cell culture applications. Explore the protocol library to find the protocol for the cell combination of interest for your application. If your combination of interest is not covered, please contact our team.
bit.bio ioDisease Model Cells and ioTracker Cells use the same user manual as their genetically matched wild-type control cells. While CRISPR-Ready ioCells have similar culture conditions to the corresponding wild-type cell control, these products have a separate user manual that includes methods for guide RNA delivery.
bit.bio ioCells have been extensively used for multi-electrode array (MEA) assays. Refer to the protocol library for a complete range of validated protocols.
Please note: Early Access ioAstrocytes (ioEA1093) are not suitable for electrophysiological assays, such as MEA.
ioCells can be transfected with mRNA and transduced with lentivirus. bit.bio has developed and optimisedprotocols for transfection and transduction for individual cell types.
bit.bio CRISPR-Ready ioCells constitutively express Cas9 or dCas9 for CRISPR gene knockout, activation, or interference. For optimised CRISPR gene editing protocols, refer to the specific CRISPR-Ready product user manual.
For advice on designing sgRNA for CRISPR-Ready ioCells, contact our team for technical support.
Lower than expected cell counts are often the result of minor deviations in the cell thawing or counting process. To ensure accuracy, verify that thawing and counting were performed as specified in the cell type-specific user manual. Common factors affecting yield include thawing speed and the precision of the cell counting method. For a step-by-step guide on best practices for thawing ioCells, we recommend visiting our video tutorials library.
Always centrifuge the cells at the recommended speed for the specified time. Monitor the size of the cell pellet and do not disturb the pellet while aspirating the media. Follow the cell type-specific user manual to resuspend the cell pellet in the recommended volume of media and ensure the cells are fully resuspended prior to counting the cells. Perform 3 cell counts for accuracy and refer to the ioCells counting protocol and workshop for guidance.
If lower than expected counts occur across more than one vial, note the lot number(s) and contact our technical support team.
Cell viability is highly dependent on counting methodology and thawing technique. Ensure that the cells were properly stored and thawed according according to the cell type-specific user manual. ioCells should be stored in liquid nitrogen or -150°C immediately upon receipt. The cryovial should be removed from liquid nitrogen immediately before cell thawing and kept on dry ice. Remove the cryovial from dry ice and immediately immerse into a 37°C water bath - the use of a bead bath is not recommended and may impact cell viability.
bit.bio recommends thawing no more than 3 vials at once per handler to reduce time in suspension. Refer to the cell type-specific user manual for required medium temperatures during cell collection and resuspension following centrifugation to prevent thermal shock.
If strict adherence to the cell type-specific user manual was maintained, note the viability and lot number(s) and contact our technical support team.
If your bit.bio ioCells do not recover after thawing, ensure you are following the instructions in the user manual carefully, and that the thawing process is carried out as recommended. Typical reasons for impaired recovery post-thawing include improper storage and not using a water bath at 37°C to thaw ioCells. Ensure that ioCells are stored in liquid nitrogen or at -150°C and transferred to a 37°C water bath immediately to thaw.
If issues persist, we recommend taking clear images of the cells at 4X and 10X magnifications and contacting our technical support team.
If your bit.bio ioCells are not adhering or are detaching from the plates, first ensure that the correct substrate or coating is being used, as specified in the cell type-specific user manual. Always use freshly prepared coating solution; note that pre-coated plates are not recommended for bit.bio cells.
If the user manual recommends coating in Poly-D-Lysine (PDL) made up in borate buffer, ensure that any excess PDL-borate has been sufficiently rinsed and completely dried prior to cell seeding, as the high pH of borate in the coating solution is toxic to cells.
It is important to avoid disturbing the plates after seeding, as movement can interfere with proper attachment. When performing medium changes, slowly dispense the medium in a dropwise manner against the wall of the well. bit.bio recommends a controlled dispensing speed of 1 mL over 20 seconds.
If issues persist, we recommend taking clear images of the cells at 4X and 10X magnifications and contacting our technical support team.
If you are observing clumping of your ioCells, first ensure that strict adherence to the coating procedure in the cell type-specific user manual was followed. bit.bio recommends the resuspension of Poly-D-Lysine (PDL) in borate buffer for the PDL coating solution. Ensure fresh borate buffer is used at the recommended pH. Follow the user manual instructions for incubation with the coating solution, and washing and drying steps. When coating with Geltrex Flex, always ensure that the Geltrex Flex is kept at 4°C as it will begin to polymerise at temperatures above 4°C.
If issues persist, take clear images of the cells at 4X and 10X magnifications and contact our technical support team.
Cell morphology is an indicator of cell health and maturity. If you have questions regarding your bit.bio cell morphology, refer to the product-specific pages or the cell type-specific user manuals for reference images. If questions remain, take clear images of the cells at 4X and 10X magnification and contacting our technical support team.
Cell morphology is a primary indicator of cell health and maturity. If you have concerns regarding cell appearance, first consult our product-specific pages or the cell type-specific user manuals for reference images of healthy cultures at various stages. Most health issues stem from deviations in reagent handling. Ensure strict adherence to reagent preparation and storage. Medium must be pre-warmed in a 37°C water bath and added gently to avoid mechanical stress, following the recommended feeding schedule. If questions remain, take clear images of the cells at 4X and 10X magnification and contact our technical support team.
To prevent contamination, always follow standard aseptic techniques, including working within a properly maintained laminar flow hood and ensuring that the hood and any materials entering the workspace are sprayed with 70% ethanol. Using fresh, sterile media and reagents is essential for maintaining a contamination-free environment.
bit.bio ioCells cannot be treated with antibiotics once contaminated. When culturing ioCells, practice standard aseptic techniques in a properly maintained laminar flow hood and wear a clean lab coat and gloves. Ensure the hood and any materials entering it are sprayed with 70% ethanol and use fresh, sterile media and reagents.
If your ioCells have recovered after thawing, but are dying after days in culture, review and ensure strict adherence to the cell type-specific user manual. Most cell viability issues stem from deviations in reagent handling and storage and media preparation and changes. Prepare media fresh on the day of feeds with reagents at the recommended concentrations. Pre-warm the media to 37°C in a water bath and add gently to avoid mechanical stress. bit.bio recommends a controlled dispensing speed of 1 mL over 20 seconds. If performing full (100%) media changes, ensure that the cells do not dry out and do not keep the plate outside of the incubator for an extended period of time as this can result in cell death.
If questions remain, take clear images of the cells at 4X and 10X magnification and contact our technical support team.
If you are experiencing issues with immunocytochemistry (ICC) antibody staining, consult bit.bio's ICC protocols and troubleshooting page for step-by-step guidance on how to stain the cells with a list of validated antibodies routinely used at bit.bio. While antibodies from other vendors or for different markers may be compatible, only the antibodies listed in the protocols have been validated by bit.bio.
ioCells undergo rigorous phenotypic characterisation, and a list of reference markers is provided per cell type. If you are not seeing the expected signal for a specific marker, consult our ICC protocols and troubleshooting page for step-by-step guidance on how to stain every cell type with a list of validated antibodies routinely used at bit.bio. While antibodies from other vendors or for different markers may be compatible, they should be tested and optimised; we cannot guarantee that alternative reagents will perform to the same specifications as those tested by bit.bio.
bit.bio cells are highly suitable for multi-cell culture applications; validated protocols for specific cell combinations are available in our protocol library. Within a protocol, wild-type and disease model cells of the same cell type can be interchanged. If your combination of interest is not yet covered, please contact our team.
Note that substituting cell types within established protocols will require careful optimisation. Key parameters, including seeding order, seeding timing, seeding densities, and surface coatings, may need adjustment, and the cross-compatibility of individual medium components should be thoroughly evaluated.
If your question is not covered here, please contact our team directly.