Human midbrain dopaminergic progenitors

Abstract Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. “Human midbrain dopaminergic progenitor” jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.

The following content constitutes indispensable articles of this standard through normative reference.For dated references, only the edition cited applies.For undated references, only the latest edition (including all amendments) applies: • Ws 213 Diagnosis of hepatitis C.
• Pharmacopoeia of the People's Republic of China (2020).
• National Guide to Clinical Laboratory Procedures.

| TERMS AND DEFINITIONS
The following terms and definitions apply to this document.

| Human midbrain dopaminergic progenitors
The neural progenitor cells that can differentiated into dopaminergic neurons with dopamine synthesis and release capacity.

| Midbrain
The middle portion of the future brain in vertebrate neural tube that connects the diencephalon and the hindbrain.
Note: This region gives rise to superior and inferior colliculus, substantia nigra and red nucleus.

| Dopamine
A type of neurotransmitter, the precursor of norepinephrine and epinephrine, a key neurotransmitter decreased in Parkinson's disease brain.

| ABBREVIATIONS
The following abbreviations are applicable for this document: 3. Organizations conducting cell research and/or production shall establish and implement standards for cell donor evaluation, collection methods, transportation, handover and storage to ensure the safety of donors and cells.The source material shall be accompanied with detailed documentation on the acquisition methods and donor information, including but not limited to the donor's general information, past medical history and family history.The information of donor's blood type (e.g., ABO, Rh) and human leucocyte antigen alleles shall be documented as necessary.
4. The origin of cells shall be traceable by referring to the relevant informed consent and/or their genomic and functional analysis data.
5. Ancillary materials such as culture medium and growth factors shall meet the corresponding quality control requirements.The ancillary materials can be inspected and tested if necessary.6.When using animal serum, they shall be free of animal-derived viral contamination.Serum from animals in geographical regions with prion epidemics (e.g.bovine spongiform encephalopathy) shall be prohibited.
7. The donors shall be screened for HIV, HBV, HCV, HTLV, EBV, HCMV and TP, and the results shall be documented.

| Cell morphology
mDAPs under adherent culture exhibit irregular shapes; the nucleus of mDAP is indistinguishable under a microscope.

| Chromosome karyotype
The karyotype shall be normal as 46, XX or 46, XY.

| Cell markers
The double-positive rate of FOXA2 and LMX1A shall be ≥70.0%,and the double-positive rate of LMX1A and EN1 shall be ≥15%.

| Functional characterization
mDAPs shall be able to further differentiate into dopaminergic neurons, which has the following characteristics: Cell morphology: some neurons aggregate and shall have extended nerve fibres.

| Differentiation
1.The original cells, equipment, culture systems and operation procedures used for cell differentiation shall be defined and documented.The standard operating procedure for cell differentiation shall be established for reproducibility.
2. The differentiated cells shall be clearly defined by characteristics, including but not limited to morphology and marker gene expression.

| Cryopreservation
1. Cryopreserved cells shall be recorded with the cell name, culture conditions, passage number, operator's name and freezing date, and so on.
2. The cryopreservation procedure shall follow the known principles of cell cryopreservation and shall be recorded.

| Thawing
1.The thawing process shall be as rapid as possible to ensure the cell viability and biological activity.
2. Cell information shall be labelled including but not limited to the cell name, passage number, culture conditions, operator's name or initials, thawing date and time.

| Cell STR identification
The STR signature of mDAPs shall be consistent with that of original donor cells.

| Cell morphology
Observe the morphology of cells using an optical microscope.

| Chromosome karyotype
The method in the Pharmacopoeia of the People's Republic of China (2020) shall be followed.

| Cell viability
The method in Annex A shall be followed.

| Cell markers
The method in Annex B or flow cytometry analysis method shall be followed.

| Functional characterization
The method in Annex C shall be followed.

| Fungi
The "1101 Sterility Inspection Method" in Pharmacopoeia of the People's Republic of China (2020) shall be followed.

| Bacteria
The "1101 Sterility Inspection Method" in Pharmacopoeia of the People's Republic of China (2020) shall be followed.

| Mycoplasma
The "3301 Mycoplasma Inspection Method" in Pharmacopoeia of the People's Republic of China (2020) shall be followed.

| Human immunodeficiency virus
The nucleic acid test method in WS 293 shall be followed.

| Hepatitis B virus
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| Hepatitis C virus
The nucleic acid test method in WS 213 shall be followed.

| Human T-lymphotropic virus
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| Epstein-Barr virus
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| Human cytomegalovirus
The nucleic acid test method in National Guide to Clinical Laboratory Procedures shall be followed.

| Treponema pallidum
The nucleic acid test method in WS 273 shall be followed.

| INSPECTION RULES
7.1 | Sampling method 1.Cells produced from the same production cycle, same production line, same source, same passage and same method are considered to be the same batch.
2. Three smallest units of packaging shall be randomly sampled from the same batch.

| Quality inspection and release
1.Each batch of products shall be subject to the qualify inspection before release, and inspection reports shall be attached.
2. The quality inspection items shall include all the attributes specified in 5.2.

| Review inspection
Review inspection shall be performed by professional cytological testing institutions or laboratories as necessary.

| Decision rules
1. Products that pass all requirements in 5.2 for the quality inspection for release are considered to be qualified.Products that fail to pass one or more requirements in 5.2 for the quality inspection for release are considered to be unqualified.
2. Products that pass all requirements in 5.2 for the quality review inspection are considered to be qualified.Products that fail to pass one or more requirements in 5.2 for the review inspection are considered to be unqualified.

| INSTRUCTIONS FOR USAGE
The instructions for usage shall include, but not limited to:  The relationship between the peak area and the dopamine concentration of the sample is calculated according to Equation (C1): where y is peak area of dopamine standards, k is the conversion factor, x is concentration of dopamine standards and b is constant.
The dopamine release is calculated according to Equation (C2): where R is dopamine release, X is dopamine concentration of cell samples, v is volume of KCl solution used to treat cell samples and n is total number of treated cells.

EBV
cytomegalovirus HIV: human immunodeficiency virus HTLV: human T-lymphotropic virus mDAP: midbrain dopaminergic progenitor STR: short tandem repeats TP: Treponema pallidum 5 | TECHNICAL REQUIREMENTS 5.1 | Raw materials and ancillary materials 1.The collection of raw materials shall be in accordance with the domestically legal and ethical requirements.2. Valid informed consent shall be signed by the donor in writing.The content of informed consent shall include but not limited to the research purpose, potential research and clinical applications under appropriate conditions, feedback on unexpected discoveries, potential commercial value and other issues affiliated to the research.Mechanisms for protecting the personal data and privacy of donors shall be established.
that need attention.Note: Provide endotoxin content according to user's requirement.
include but not limited to: a. product name; b. cell number; c. lot number; d. production organization; e. production date.C.4.5.| Analysis of results