Amniotic Fluid Cells Biobank for Research on Fetal Mesenchymal Stem Cells

ethical principles. Collateral biobanks are present to collect, storage and manage donor sera and DNA samples, to perform microbiological and genetic tests necessary for the unit release.


Project description
The research Biobank (BB) "Anna Maria Ferraro Cutino", Hematology for Rare Diseases of Blood and Hematopoietic Organs, AOR-Villa Sofia-Cervello, started in 2010 by a Grant of the Sicilian Department of Health for the improvement of Sicilian Cell and Tissue Banks.
Within the BB are collected, characterized, stored and distributed amniotic fluids (AF) from amniocentesis performed for maternal age. Donors are between the ages of 36 and 45 and they have no positive history for genetic diseases.
Women choose to donate to the BB the first 3 ml of AF that cannot be used for prenatal diagnosis and would therefore be eliminated.
The AF BB research project has been approved by the local ethics committee (prot. 128.01/12/2010).
Since 2014, AF BB is part of the Integrated Clinical-Biological Network for Regenerative Medicine (RIMEDRI) through an interactive platform realized by a Grant of Sicilian Productive Assets Department (http://rimedri. ospedaliriunitipalermo.it).
In 2016 AF BB joined BBMRI-ERIC Italian Node (http:// www.bbmri.it/home) with the name of BCSMF: bank of mesenchymal fetal stem cells.
AF are rich in fetal mesenchymal stem cells (FMSC) that are able to regenerate tissues (adipose, cartilage, bone), have anti-inflammatory and immunosuppressive properties, and a high proliferative potential [1].
For this reason, BCSMF is an important resource of FMSC that could be employed in regenerative medicine [2], autoimmune and chronic inflammatory diseases, allogenic bone marrow transplantation, in-utero transplantation [3].
The BCSM is a research biobank and AF samples can be used for research only.
The main goals of biobank include: -collection, storage and management of AF samples and data, -collection, storage and management of donor sera and DNAs, -sharing AF samples and data with the scientific community, -sharing protocols and know-how [4].
To date, BCSMF has collected and stored 300 AF and related clinical data.

Species
Human.

Classification 2
BCSMF collects, characterizes and cryopreserves AF samples and related clinical data. AF donor sera and DNAs of AFs are also stored.

Temporal coverage
From 2010 to present, on-going with no fixed expiry date.
Steps AF donors, who undergo amniocentesis for maternal age, are selected by a gynecologist of Prenatal Diagnosis Unit, who shows the biobank aims and provides donation informed consent.
Samples are transfer in a refrigerated CE container keeping transport temperature between 10°-20°C; time between sampling and treatment is 30'.
For cryopreservation DMSO is directly added to the AF cell suspension at a 10% final concentration.
Samples are aseptically treated under a dedicated biobank laboratory A+ laminar flow hoods.
AF are frozen in a controlled rate freezer according to a profile suitable for stem cells, up to a final temperature of −140°C, and stored in nitrogen vapor dedicated dewars at −175°C.
AF underwent aerobic and anaerobic contamination test through seeding in specific media.
Integrity of thawing AF cells is tested with the trypan blue method, the found vitality is 70%.
Donor serum is obtained by centrifugation at 1500g for 10'; two aliquots of 1ml are stored in −40°C biobank freezer with continuous monitored temperature and data logger.
Donor DNA is extracted from peripheral blood by phenol-chloroform method; quantification and purity evaluation are performed on spectrophotometer. DNA integrity is checked by 1% agarose gel sybersafe stained, in comparison with Lambda DNA marker. DNA is dissolved in sterile distilled water and stored in the −40°C biobanks freezer.
Prenatal Diagnosis Unit provides donor clinical data; microbiology laboratory of AOR Villa Sofia-Cervello provides donor virology and microbiological AF test for aerobic and anaerobic agents.
Samples encoded data are recorded in an Excell database. AF modules are scanned and stored on a biobank dedicated PC and on external safe informatics support whose access is limited to biobank manager and staff. All folders are stored in limited access fireproof cabinets.
FA cells, both fresh and after thawing, were cultured in MSC expansion serum free medium (STEM CELL-Voden); and after 8 days of culture they developed CFU-F colonies. At 15 days culture there was fibroblast growth, which became confluent at 28 days.
On expanded AF-FMSC, both fresh and after thawing, CD106, CD146 and CD105 mesenchymal markers, and CD45 and CD34 non-mesenchymal markers were evaluated, by flowcytometry analysis.
The flowcytometry analysis of expanded FA-MSC, both from fresh and after thawing, revealed the presence of mesenchymal markers CD146 and CD105 and the absence of CD34 and CD45 hematopoietic/endothelial markers.
The fetal origin of expanded AF-FMSC, both from fresh and after thawing, was verified and confirmed by QF-PCR on DNA from expanded AF-FMSC and maternal mononucleates.
Stam potential of AF-FMSC, both from fresh and after thawing, was evaluated, before and after expansion, by expression analysis (RT-PCR) of Oct4 and Nanog genes. As control, cDNA from maternal peripheral blood was used.
The specific RT-PCR performed on the expanded AF-MSC, both from fresh and after thawing, cDNA revealed the presence of both Oct4 and Nanog expression.
-Peripheral blood in EDTA (for DNA extraction and serum collection).

Type of long-term preservation
-Frozen at −40°C (for DNA and serum preservation), DMSO and liquid nitrogen for AF.
All samples are stored in temperature-monitored freezers that are locked and accessible only by BCSMF staff. In progress.

Bioresource type
Human amniotic fluid biobank.

Type of sampling
Amniotic fluids (AF) from diagnostic amniocentesis.

Anatomical site
Amniotic cavity.

Disease status of patients/source
Patients who undergo amniocentesis performed for maternal age.

Clinical characteristics of patients/source
-General data.
-Data on pregnancy (age and reason of amniocentesis).

Size of the bioresource
To date, 300 samples of amniotic fluid have been collected and stored.

Vital state of patients/source
All patients are alive at sampling.

Clinical diagnosis of patients/source
Not clinical features, amniocentesis performed for maternal age.

Control samples
NA.

Release date
NA.

Access criteria
Access to database is restricted to limited number of BB member staff. The access rules for BCSMF samples are the following: -The biological specimens can be distributed only for research and only if in the BB two aliquots of the same sample are present. -The use of biological specimens must be non-profit.
-The distribution of biological specimens must be authorized by the biobank manager. -In the resulting sci entific publication, the authors garantee to mention the source of the samples, to thank for the service, to send a copy of the publication. -Research project have to be in agreement with BCSMF mission, and have a wide scientific significance. -Sample distribution will be free, except for direct shipping costs.

(4) Reuse potential
Public and/or private scientific organizations, wishing to use FA samples, have to make an application on a specific form issued by the BB, to provide all the required data and to specify the purposes of the research for which they plan to employ FA samples. All requests will be submitted to the attention of biobank Evaluation Committee, consisting of biobank manager and quality manager, who verifies the interest and the impact of the project on the scientific community.
If the request is not assumed appropriate, applicants will be aware about the reasons of the refusal.