Should Must May MITreg Checklist 1) Cells at the start of procedure a) Essential information about the donor i) Species and strain Species Strain (if applicable) ii) Characteristics of the organism Health Age Treatment/Environment Individual identifier number Source of purchase (if applicable) b) Source of cell material Organ, tissue, fluid or blood product Source (if applicable) Quantity (volume, size or weight) Anti-coagulant (if applicable) If using cryopreserved sample: Method and duration of storage Initial cell counts Ethical committee approval/ written informed consent c) Cell separation process i) Cell handling and labelling Cell extraction method Tissue conditions between tissue retrieval and cell separation Duration Temperature Container Fluid Cell labelling Buffers and reagents (incl. source) Cell suspension volume and concentration Incubation temperature and duration Washing steps ii) Cell separation equipment and process Methodology Equipment Presence of target cells in starting material described Should Must May d) Phenotype For any of the below, indicate the percentage of cells displaying the characteristic (if known) i) Cell surface and intracellular markers Molecules measured (using CD names) Details of reagents used and source (incl. mAb clone, fluorochrome) Methodology Stimulus and time of stimulation (if applicable) Gating strategy to determine positive cells ii) Secreted molecules Molecules measured Details of reagents used (incl. mAb clone, conjugate) and source Methodology Cell density/ml of medium and type of tissue culture plate Time point of supernatant collection Stimulus and time of stimulation (if applicable) Hi) Epigenetic modifications Epigenetic modification relevant to the characteristics iv) Specificity Specificity of the cells (polyclonal or antigen-specific) Methodology used to obtain specificity Methodology used to confirm specificity e) Cell numbers i) Absolute cell number Total number of cells at the end of the isolation process Methodology ii) Viability Percentage of viable cells Methodology 2. Expansion/Differentiation a) Pre-culture conditions Storage conditions Fluid Type of container Temperature Fresh or thawed Storage time Should Must May b) Culture conditions i) Cell number The total number of cells put into culture ii) Cell concentration The number of cells per ml of medium at start of culture Hi) Culture medium Type(s) of medium Source(s) Additives (excluding agents to maintain/induce Tregs) Refreshment of the medium iv) Culture container Type of container Size Manufacturer Cell culture volume per container or well Total number of containers or wells v) Culture environment Temperature and C02 concentration Use of pre-warmed medium Equipment c) Differentiation/tolerization protocol Name of cytokine(s) or other agent(s) used Concentrations Time-point(s) added to cell culture Total length of the culture period Rounds of stimulation Number of cell splitting d) Stimulus Polyclonal/antigen-specific/allo-antigen Stimulus (agent and/or accessory cell) Source Concentration Time point(s) added to culture Restimulation conditions (if applicable) e) Storage Storage time Storage conditions If fresh Fluid Container Temperature If cryopreserved May Must Should Freezing/thawing process Freezing medium Cell recovery & viability after thawing Time point at which cells are stored if different to the end of the culture process 3. Cells after expansion/differentiation a) Phenotype For any of the below, indicate the percentage of cells displaying the characteristic (if known) Stability of the phenotype (if tested) Phenotype tested on fresh or thawed cells i) Cell surface and intracellular markers Molecules measured (using CD names) Details of reagents used and source Methodology Stimulus and time of stimulation (if applicable) Gating strategy to determine positive cells ii) Secreted molecules Molecules measured Details of reagents used and source Methodology Cell density/ml of medium and type of tissue culture plate Time point of supernatant collection Stimulus and time of stimulation (if applicable) Hi) Epigenetic modifications Epigenetic modification relevant to the characteristics b) Functional assay Response of the cells to a defined stimulus Behaviour of other biological entities after exposure to the cells If using accessory cells, describe phenotype and source c) Cell numbers i) Absolute cell number Total number of cells at the end of the isolation process Methodology ii) Viability Percentage of viable cells Methodology Should Must May d) Dosing Dose of cells transferred into organism (if applicable) Vehicle (solvent/medium) and intermediate components (for clinical trials only) e) Quality control (for clinical trial only) Specificity Purity Sterility Potency 4. About the protocol a) Regulatory authority External authority that approved the protocol Does protocol follow GMP? □ □ □ b) Purpose The disorder for which the cell treatment has been manufactured c) Relationship between the source organism for the cells and the target organism Allogeneic/Autologous/ Xenogeneic/Syngeneic d) Contact details Name and contact information of the corresponding author(s) e) Citation Acknowledge the MITREG reporting guidelines