Introduction
Research into fetal brain tissue holds immense significance for neuroscience and the elucidation of disease mechanisms[
1]. Given that the fetal brain is in a phase of rapid development, key processes like neurogenesis, cell migration, and synaptic plasticity are pivotal for comprehending normal brain development as well as its deviations[
2,
3]. There exists a pressing demand to devise a standardized operational protocol for China’s fetal brain tissue bank. This initiative will not only bridge the existing gap in China’s standardized framework for fetal brain tissue research, thereby bolstering fundamental neuroscience research on brain development physiology and pathology, along with clinical disease mechanism exploration, but also aid brain bank developers in navigating and overcoming the dual hurdles of ethical standards and sample quality control.
Currently, the construction of human brain tissue banks in China has not extended to the realm of fetal brain tissue. Domestically, the “Standardized Operational Protocol for the China Human Brain Bank Consortium (2nd Edition),” issued by the “Chinese Consortium for Human Brain Banking,” mainly outlines the comprehensive procedural norms for adult brain tissue collection, encompassing requirements for pertinent ethical review documents, clinical data acquisition, sampling procedures, processing and storage techniques, and pathological diagnosis[
4]. Nevertheless, owing to the distinctive developmental stage of fetal brain tissue—characterized by notable disparities in processes such as neural stem cell proliferation and synapse formation compared to adults—and the challenges associated with sample procurement, a dedicated standardized operational system for the establishment of fetal brain tissue banks remains to be formulated.
The creation of a standardized operational protocol for fetal brain tissue banks will guarantee uniformity in sampling locations, fixation techniques, and preservation conditions, thereby minimizing experimental discrepancies stemming from operational variations. This will enable fetal brain tissue samples to serve as effective and vital specimens for research into the early pathogenesis of diseases, including neurodevelopmental disorders. Furthermore, it will contribute to elevating China’s standing and competitiveness in the global neuroscience arena.
This Standardized Operational Protocol (SOP) is an enhancement and expansion of the “Standardized Operational Protocol for the China Human Brain Bank Consortium (2nd Edition)”[
4]. It draws on the practical experiences accumulated by numerous domestic brain banks and, in a pioneering move, incorporates post-mortem fetal brain
ex vivo Magnetic Resonance Imaging (MRI) technology as an optional supplementary evaluation tool[
5]. The primary objective of this protocol is to set forth unified and standardized operational guidelines for fetal brain and spinal cord sample banks. It aims to reinforce the ethical acquisition of samples, ensure their standardized processing, and facilitate open sharing. Additionally, the protocol provides technical backing for the efficient procurement of fetal brain and spinal cord samples that meet research criteria, thereby fostering the innovative advancement of brain science research in China.
Materials and methods
Objectives of fetal brain tissue bank collection
The creation of a fetal brain tissue bank is geared towards offering essential resource backing for research in neurodevelopmental biology, medical education endeavors, and the advancement of diagnostic and therapeutic approaches for neurological conditions. Its foremost objective is to unravel the pivotal mechanisms underlying the development of the fetal nervous system and to probe into the early beginnings of congenital neurological diseases. Although China has witnessed notable enhancements in prenatal care accessibility, there are still constraints in the early detection and conclusive diagnosis of structural or functional irregularities within the fetal nervous system. To tackle this challenge, the existing fetal brain tissue bank employs a wide-ranging collection strategy. It amalgamates clinical data from expectant mothers and carries out postmortem pathological examinations of fetal brain tissue. Through this method, a thorough database system is established, which includes resources of both typically developing and anomalous fetal brain tissues across a spectrum of conditions.
General objectives of fetal brain tissue bank collection
(1) The bank aims to acquire fetal brain tissue spanning different gestational ages, genders, familial genetic backgrounds, and histories of environmental exposures during pregnancy.
(2) The bank shall collect brain tissue from both normally developing fetuses and those with neurological abnormalities, including but not limited to: congenital brain malformations, nervous system tumors, neurodevelopmental disorders, hereditary neurological diseases (e.g., Down syndrome), and brain injuries caused by maternal infections (e.g., cytomegalovirus) or toxins (e.g., alcohol) during pregnancy[
6].
(3) For fetuses with a family history of hereditary neurological diseases, the bank shall collect pedigree samples and retain parental peripheral blood specimens to provide complete resources for studying genetic patterns and mechanisms.
(4) For fetuses with multisystem abnormalities (e.g., Walker-Warburg syndrome[
7] and Meckel-Gruber syndrome[
8]), the bank shall implement a coordinated multi-organ collection strategy to preserve affected tissues and construct integrated resources linking phenotypic, tissue, and genetic information.
(5) The bank shall establish a comprehensive sample identification, classification, and information management system to ensure traceable origins, accurate data, scientific categorization, and controlled usage of all samples.
(6) It is essential to strictly control the interval between fetal demise and tissue processing, optimize preservation protocols, and maintain brain tissue integrity and biomolecular stability to minimize sample degradation.
Targeted collection for specific research projects
Alliance members may conduct targeted collection of samples for specific neurological diseases in fetuses, along with matched control brain tissue, based on research priorities:
(1) It is essential to clearly define disease types and simultaneously collect corresponding normal control brain tissue.
(2) The bank shall record key information during collection, including fetal abnormalities during pregnancy, diagnostic evidence, maternal exposure history, and familial genetic history.
(3) The bank shall preserve samples according to unified standards after neuropathological confirmation. Disease categories include, but are not limited to congenital brain malformations, nervous system tumors, neurodevelopmental disorders, hereditary neurological diseases, brain injuries caused by pregnancy-related teratogenic factors, systemic diseases with cerebral developmental abnormalities, and mixed cases[
6].
Legal basis and ethical review
Legal framework
The establishment and operation of China’s fetal brain tissue bank must comply with national laws and regulations, including the Civil Code of the People’s Republic of China (2021), Biosafety Law of the People’s Republic of China (2021), the Regulations on the Administration of Human Genetic Resources of the People’s Republic of China (2019, revised in 2024), the Law of the People’s Republic of China on Prevention and Control of Infectious Diseases (1989, revised in 2025), and ethical guidelines for human organoid research (2025).
Ethical review principles
(1) Informed consent: The bank shall respect and protect the autonomy of donors or their legal representatives in deciding whether to participate in research. The hospital shall fully inform pregnant women and their families about the use of samples, processing methods, and potential risks before collecting fetal brain tissue. Pregnant women must sign the Informed Consent Form for Biological Sample or Clinical Information Donation during hospitalization.
(2) Privacy protection: The bank shall safeguard the privacy of pregnant women by transparently communicating the storage, usage, and confidentiality measures for fetal information. Unauthorized disclosure of fetal or maternal data to third parties is prohibited.
(3) Non-commercialization: No organization or individual may trade or engage in disguised trading of fetal brain tissue samples collected by the bank.
(4) Benefit sharing: International collaborative research involving Chinese human genetic resources (fetal brain tissue) must adhere to principles of equality and mutual benefit, with clear agreements outlining rights and obligations among all parties.
(5) Protection of vulnerable populations: For fetal brain tissue collection involving minor pregnant women, consent must be obtained from both the minor and their legal guardian.
Ethical review process and requirements for sample applications
Applicants must submit a formal request for fetal brain tissue, which will be reviewed promptly by the bank’s academic committee for approval. Approved users must sign a fetal brain tissue usage agreement before receiving samples from the bank. Applicants must demonstrate prior academic achievements relevant to the requested tissue and secure national or provincial research funding. Projects must be approved by their institution’s ethics committee. Usage principles:
(1) Samples may not be transferred to third parties and must be used solely by the researchers listed in the application at the designated location.
(2) All accompanying data must remain confidential.
(3) Publications using fetal brain tissue must acknowledge the bank.
(4) Intellectual property derived from the use of samples belongs to the researchers.
(5) Ethical compliance in fetal brain tissue usage is mandatory.
(6) Academic outcomes must be reported annually to the bank before publication.
Sources of brain tissue
Currently, the primary source of fetal brain tissue in China is through clinical donations from pregnant and postpartum women. This process is conducted in accordance with Chinese laws and ethical principles, with pregnant women or the immediate family members of the fetus signing informed consent forms.
Collection assisted by clinical hospitals
Medical institutions such as obstetrics departments in clinical hospitals and maternal and child health care hospitals provide information on induced labor donations, which are then accepted by body donation reception stations. The donation procedure is as follows:
(1) Pregnant or postpartum women sign the “Informed Consent Form for the Donation of Biological Samples or Clinical Information” during their hospital stay.
(2) After the fetus is delivered, family members need to provide a death certificate, the informed consent form, and medical records.
(3) Immediate family members have the right to withdraw from the donation program before completion of the donation.
Popular science promotion
To expand the capacity of the fetal brain tissue bank and strengthen communication between body donation reception stations and obstetrics hospitals or maternal and child health care hospitals, popular science promotion activities are conducted based on the characteristics of each medical institution:
(1) Popular science materials: The bank shall produce promotional brochures and place them reasonably in areas designated for volunteer donation popular science promotion.
(2) Training for medical staff: Relevant personnel from the fetal brain tissue bank or clinical hospitals should possess good communication skills and sufficient knowledge background to answer donors’ questions at any time.
Clinical data collection
Relevant clinical information is provided by the healthcare institution where the pregnant woman receives care.
Basic maternal information
(1) Personal details: Age, occupation, education level, ethnicity, marital status, etc.
(2) Contact information: The bank shall record the pregnant woman’s contact details and address to facilitate follow-up and supplementary data collection.
(3) Past medical history: Chronic diseases (e.g., hypertension, diabetes, thyroid disorders), infectious diseases, and family medical history, including hereditary neurological disorders.
(4) Obstetric history: Gravidity, parity, and histories of abortion, preterm delivery, stillbirth, etc. Multiple miscarriages or preterm births may increase the risk of fetal brain abnormalities.
Pregnancy-related information
(1) Prenatal examination results: Results from routine tests (e.g., blood counts, urinalysis, liver and kidney function, blood glucose, and lipid profiles) and specialized examinations (e.g., ultrasound/MRI imaging, genetic testing reports, Down syndrome screening, non-invasive DNA testing, and amniocentesis).
(2) Medication use during pregnancy: The hospital shall document the names, dosages, and durations of all medications taken by the pregnant woman during pregnancy.
(3) Living environment during pregnancy: Environmental factors such as air quality, water quality, and exposure to harmful substances (e.g., pesticides, heavy metals, and radiation).
(4) Psychological status during pregnancy: The hospital shall evaluate the pregnant woman’s mental health, including anxiety, depression, or other emotional issues.
Fetal-related information
(1) Basic fetal details: The hospital shall record the fetal gender, estimated due date, weight, and length.
(2) Intrauterine fetal development assessment: The hospital shall record intrauterine developmental evaluations, such as fetal movement patterns and cardiotocography results. Abnormalities may indicate risks like fetal distress, which could impact brain development.
(3) Fetal disease diagnosis: If a disease is diagnosed, the hospital shall document its name, diagnosis date, method, and treatment details. Conditions like neural tube defects, hydrocephalus, or congenital cerebral palsy directly affect brain development.
Fetal delivery-related information
(1) Mode of delivery: The mode of delivery includes medical induction followed by vaginal delivery or cesarean section, or spontaneous vaginal delivery following the natural onset of labor. The latter is relatively uncommon and may occur in cases of intrauterine fetal death with spontaneous onset of uterine contractions. Vaginal delivery may cause varying degrees of mechanical injury or compression to the fetal head. Thus, during neuropathological examination, it is essential to carefully distinguish between mechanical trauma induced by vaginal delivery process from pathological changes (e.g., hemorrhage or infection) that developed in utero during fetal life. Notably, all samples were obtained from pregnancies terminated for medical indications (e.g., severe congenital anomalies and intrauterine fetal death).
(2) Time from fetal demise to delivery: The interval between intrauterine fetal death and delivery is recorded, as it is a key factor influencing the degree of tissue autolysis. Furthermore, a prolonged medical induction may accelerate autolysis of fetal brain tissue, primarily due to sustained uterine contraction pressure or changes in the intrauterine environment.
Procedures for fetal brain tissue collection
The quality of fetal brain specimens is the cornerstone of tissue banking. Precise control over collection, fixation, and the time intervals between procedural steps is critical. Staff should transport the fetal remains to the tissue collection site as soon as possible, with the optimal time for brain extraction being within 60 min after fetal death following separation from the mother. The collection site is typically a dedicated anatomical laboratory for the human brain tissue bank or a hospital pathology anatomical laboratory, or other suitable locations for tissue collection operations, and tissue collection should be completed promptly. If tissue collection cannot be performed immediately, the remains should be stored at low temperatures to minimize enzymatic degradation and protein denaturation in the brain tissue as much as possible.
Following collection, a macroscopic morphological screening is performed to exclude specimens showing severe autolysis, putrefaction, or significant mechanical damage. During sampling, immediate on-site evaluation includes observation of cerebrospinal fluid (color and clarity), brain tissue consistency (elasticity and firmness), and meningeal integrity.
After standard processing, fixed tissue sections are examined microscopically by a neuropathologist to assess key structures (e.g., cortical lamination and basal ganglia outlines) and provide a diagnosis. For frozen tissue aliquots, it is recommended to determine the RNA Integrity Number (RIN). This objective metric (scale 1−10, higher indicating better integrity) reflects the extent of RNA degradation and serves as a core parameter for evaluating molecular sample quality.
The complete workflow for fetal brain tissue collection is shown in Figure 1. Details illustrating different stages of fetal brains and the corresponding slicing or fine sampling procedures are presented in Figure 2.
The specific operational steps are as follows, the operator should:
(1) Image capture of fetal remains: Take images of the fetal frontal and dorsal body, placing a ruler and a unique sample identifier during photography to facilitate subsequent measurement of fetal brain dimensions.
(2) Scalp and skull dissection: Make a coronal incision along the line connecting the bilateral postauricular area to the vertex of the skull, fold back the scalp to expose the cranial bone. Cut the skull approximately 1 cm above the line connecting the supraorbital ridge to the external occipital protuberance, ensuring the incision is not too deep to avoid damaging brain tissue. After opening the skull, collect cerebrospinal fluid and dura mater, then freeze and store at −80 °C.
(3) Spinal column exposure: Create a longitudinal midline incision along the back, extending from the external occipital protuberance along the spinous processes to the sacrum. Strip ligaments and paraspinal muscle tissue attached to the spinous processes and vertebral plates, exposing posterior bony structures (e.g., spinous processes, vertebral plates, and transverse processes). Make a longitudinal incision through the posterior spinal wall or vertebral bodies and articular processes, then lift the vertebral plates to expose the dura mater.
(4) Spinal cord and brain removal: Sever spinal nerves externally to the spinal cord, split the dura mater along the anterior midline, and expose the spinal cord. Extract the fetal brain and spinal cord, place a ruler and unique sample identifier, and photograph from frontal and dorsal perspectives.
(5) Brain-spinal cord separation: Transect the brain and spinal cord at the anatomical site located inferior to the medulla oblongata and superior to the first cervical nerve root. Weigh the whole brain and cerebrum separately.
(6) Spinal cord processing: For fetuses ≥ 15 gestational weeks (GW), take three 0.5-cm segments (cervical, thoracic, and lumbar) for freezing; fix the remainder in formalin. For fetuses < 15 GW, freeze the entire spinal cord. For cases requiring disease diagnosis, place directly in formalin.
(7) Brainstem and medulla oblongata: Split the brainstem along the midline into left and right halves. Fix the right half in formalin and freeze the left half. Divide the left brainstem into four equal sections for sampling.
(8) Cerebral hemisphere separation: Separate the fetal brain into left and right hemispheres. Freeze the left hemisphere and fix the right hemisphere in formalin.
(9) Left hemisphere sampling (Figure 2): For fetuses < 15 GW, freeze directly. For special sampling needs, immerse brain tissue in artificial cerebrospinal fluid (ACSF) to minimize damage and improve efficiency. For fetuses 15−24 GW, immerse in ACSF before sampling, cutting 1 cm coronal sections sequentially from anterior to posterior. For fetusfor ≥ 24 GW, sample directly, cutting 1 cm coronal sections sequentially from anterior to posterior.
(10) Right hemisphere sampling (Figure 2): Fix in formalin for 2 weeks. For fetuses < 20 GW, cut coronal sections sequentially from anterior to posterior. For fetusfor ≥ 20 GW, isolate the following tissue blocks following specified collection standards:
(a) Optic chiasm
(b) Frontal cortex
(c) Visual cortex
(d) Mammillary body + third ventricle
(e) Putamen and globus pallidus
(f) Bilateral ventricular angles (including corpus callosum, corona radiata, cortex, and caudate nucleus)
(g) Hippocampus
(h) Midbrain
(i) Pons
(j) Medulla oblongata
(k) Choroid plexus
(l) Cerebellar hemisphere
(11) Sample preservation: Freeze all frozen samples rapidly in liquid nitrogen, then transfer to −80 °C for long-term storage. For molecular genetics testing, retain a small tissue fragment for freezing. Seal residual tissue (excluding fixed samples) in formalin, replacing the solution every 2 years.
Pathological diagnosis of fetal brain tissue
(1) Dehydration and paraffin embedding of fixed tissue samples. Utilize an automated dehydration system with the following procedure:
(a) Formalin, 12 h, 37 °C
(b) Deionized water, 0.5 h, 45 °C
(c) 70% ethanol, 4 h, 45 °C
(d) 85% ethanol, 4 h, 45 °C
(e) 95% ethanol I, 4 h, 45 °C
(f) 95% ethanol II, 4 h, 45 °C
(g) 100% ethanol I, 4 h, 45 °C
(h) 100% ethanol II, 4 h, 45 °C
(i) Xylene I, 2 h, 45 °C
(j) Xylene II, 2 h, 45 °C
(k) Paraffin I, 1 h, 62 °C
(l) Paraffin II, 2 h, 62 °C
(m) Paraffin III, 12 h, 62 °C
(2) Sectioning: Routine sectioning at a thickness of 6 μm is recommended. After mounting, place slides in a 65 °C oven overnight.
(3) Staining: Routine staining includes Hematoxylin-Eosin (HE), Nissl, and Luxol Fast Blue-Periodic Acid-Schiff (Luxol-PAS). Additional immunohistochemical staining may be conducted as required[
6]. HE staining provides an overall morphological overview, enabling the observation of the basic tissue structure, nuclear morphology, and cytoplasmic characteristics. Nissl staining specifically reveals Nissl bodies within neurons, facilitating the assessment of neuronal distribution, density, and the laminar structure of the cerebral cortex. For fetal brain samples with a gestational age of ≥ 20 weeks, Luxol-PAS staining is added to evaluate the potential for early myelination. For fetal brain samples in the late stages of pregnancy, Reelin staining can be added to assess key pathways involved in cortical neuronal migration. Additionally, glial fibrillary acidic protein (GFAP) staining can be used to evaluate the distribution and reactivity of astrocytes, while cluster of differentiation 68 (CD68) staining marks microglia to screen for inflammation and injury.
Preservation and management of fetal brain tissue-related information
The fetal brain tissue bank is responsible for maintaining confidentiality of donor family and fetal information to prevent data breaches. The bank shall:
(1) Establish electronic and retain paper archives for basic and clinical data of pregnant women and fetuses.
(2) Assign unique identification numbers to fetal brain tissues immediately upon storage and link corresponding information.
(3) Collect imaging data, including frontal and dorsal photographs of fetal brains and spinal cords, as well as section images from sampling.
(4) Capture image data from fetal brain tissue sections stained for pathological diagnosis.
(5) Maintain electronic and paper archives of pathological diagnosis reports for fetal brain tissues.
(6) Collect genetic information (e.g., RNA) and omics database data from fetal brain tissues.
(7) Gather feedback from applicants for fetal brain tissue samples.
Application and feedback for the use of fetal brain tissue
The use of fetal brain tissue requires a formal application from the user, which will be reviewed by the Ethics Review Committee and the Management Committee of the Human Brain Tissue Bank to determine whether to grant the sample. Upon approval, the user signs a fetal brain tissue usage agreement, after which the fetal brain tissue bank provides the sample to the applicant. Applicants for the use of fetal brain tissue should adhere to the following principles:
(1) Fetal brain tissue cannot be provided to third parties and shall only be used by the researchers listed in the application at the designated location.
(2) Any information provided alongside the fetal brain tissue must be kept confidential.
(3) When publishing articles using fetal brain tissue materials, acknowledgment should be given to the fetal brain tissue bank.
(4) Intellectual property rights derived from the use of fetal brain tissue materials belong to the researchers.
(5) The usage agreement requires the signatures of all participating researchers, representatives from their respective institutions, and the head of the fetal brain tissue bank.
(6) Any findings obtained by researchers using fetal brain tissue must be fed back to the fetal brain tissue bank.
Results
This standardized operational protocol specifies the framework for the establishment of a fetal brain tissue bank in China. Key outcomes are as followed:
(1) The protocol defines the collection objectives and strategies. For general collection, it establishes criteria for acquiring samples across different gestational ages, genders, genetic backgrounds and etc. For targeted collection, it provides guidelines based on research priorities.
(2) The protocol establishes a comprehensive legal and ethical framework, incorporating relevant national laws and regulations. It also defines the ethical review process for sample applications, requiring that all research projects obtain approval from their institutional ethics committee.
(3) The protocol identifies clinical donations as the primary source of fetal brain tissue in China, requiring that pregnant women or the immediate family members of the fetus sign informed consent forms prior to donation.
(4) The protocol requires detailed clinical information collection, thereby ensuring comprehensive phenotyping for subsequent research.
(5) The protocol provides detailed steps for the entire collection process, including: craniotomy, cerebrospinal fluid collection, brain and spinal cord extraction, brain-spinal cord dissection, among others. Gestational age-specific handling procedures are specified for both left hemisphere (fresh/frozen processing) and right hemisphere (formalin fixation), with detailed regional sampling guidelines for fetuses ≥ 20 GW.
(6) The protocol establishes standardized protocols for tissue embedding, sectioning, and staining, and accommodates additional immunohistochemical staining as required for specific research questions.
(7) The protocol mandates that the fetal brain tissue bank is responsible for maintaining confidentiality of donor family and fetal information to prevent data breaches.
(8) The protocol establishes principles for sample application and feedback on the use of fetal brain tissue.
Discussion
By integrating ethical oversight, clinical data acquisition, standardized sampling procedures, and pathological diagnosis, this protocol ensures operational consistency and minimizes technical variability. This implementation will enhance the reproducibility and comparability of research outcomes, facilitate collaborative research across institutes, and thereby enable reliable investigations into normal brain development and the early pathogenesis of neurodevelopmental disorders.
The establishment of our protocol aligns with and draws inspiration from key international fetal tissue biobanking. A notable example is the Dutch Fetal Biobank, which provides a valuable contemporary framework. As detailed by Dawood
et al. in 2023[
9], based on their experience with the first 329 donated specimens, this initiative emphasizes a standardized workflow from donation to storage and addresses the complex legal and ethical aspects integral to prenatal tissue collection. Beyond Europe, the Human Developmental Biology Resource (HDBR) in the UK represents a long-standing and comprehensive model[
10]. It systematically collects embryonic and fetal material and has made a transformative impact by creating the HDBR Atlas, which integrates three-dimensional models, high resolution histology sections and gene expression data[
11]. Despite these international advancements, fetal brain tissue banking in China has faced several challenges, including the absence of a unified standard, inconsistent integration of clinical and genetic data with tissue samples, and underdeveloped mechanisms for sample and data sharing. By providing a systematic framework, our protocol is designed to directly address these limitations.
Looking forward, the widespread adoption and continued evolution of this standardized protocol will catalyze transformative advances in developmental neuroscience. First, the integration of emerging technologies, such as spatial transcriptomics, will substantially expand the scientific yield of collected specimens. The incorporation of ex vivo MRI as a supplementary tool already establishes a foundation for correlative imaging-transcriptomic analyses, an approach that will increasingly define the future of developmental neuropathology. Second, future multi-center initiatives could systematically map spatiotemporal gene expression landscapes across diverse genetic backgrounds and environmental exposures, creating a comprehensive reference atlas of Chinese fetal brain development. At last, international data sharing will amplify global impact, as cross-population comparisons will reveal population-specific variations.
Conclusion
This standardized operational protocol establishes a unified framework for fetal brain tissue collection, processing, and sharing across institutions. Ultimately, this protocol will maximize the scientific value of China’s fetal brain tissue resources, thereby addressing a critical gap in China’s neuroscience research, and enhancing China’s competitiveness in the international scientific community.
The Author(s) 2026. This article is published by Higher Education Press at journal.hep.com.cn.
PDF
(2595KB)
