Clinical potential of human amniotic fluid stem cells

Subjects

Healthy gravidas scheduled for cesarean birth for clinical indications without medical complications or urgency and not in labor were included. Excluded were patients with bleeding, complicated or abnormal pregnancy or abnormal fetus.

Ethical approval

The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ Institutional Review Board or equivalent committee.

Sample collection

Informed consent was obtained prior to sample collection (NYU Grossman School of Medicine IRB study i15-01269, Lenox Hill Hospital Northwell Health IRB study 21–0,162). After laparotomy a small uterine incision was made at the proposed site for delivery and a sterile catheter inserted to collect the fluid into a sterile bag. After flow stopped the catheter was withdrawn, the cesarean completed and the collected fluid delivered to the cell therapy center for processing and cryostorage.

Simulating a clinical setting to test the stability of term AFSCs in normal saline

One sterile vial of 4 mL of cell therapy center processed term AFSCs stored in CryoStore™ at −80 °C was thawed at room temperature at approximately 21 °C for approximately 20 min. The cells were removed from the vial using an 18-gauge needle and 20 cc syringe with 11 mL sterile normal saline to a total volume of 15 mL. An aliquot of 100–200 µL was collected into an Eppendorf tube to assess cell viability and cell count. Viability and cell count were assessed using trypan blue staining, a hemocytometer, and light microscope. The sample was then allowed to sit for 1–3 h after the 20 min thaw. At the 1, 2, and 3 h mark the syringe was inverted five times and a 100–200 µL aliquot was taken and viability and cell count assessed.

Simulating a clinical setting to test the integrity of term AFSCs after passed through balloon catheter

In a clinical setting, the procedure would be to thaw the storage vial at room temperature and draw it up in a sterile fashion, diluted by normal saline for introduction to the patient by direct injection or through a catheter to the desired site. Therefore, we simulated clinical conditions using a 20 cc syringe and 18G hypodermic needle, and a balloon catheter used clinically for administering liquid to a tissue site in a patient.

Five mL NYBC sterile cells cryopreserved in CryoStore™ Multi-Chamber Freezing Chambers were taken from −80 °C and thawed at room temperature at approximately 20 °C. The mini chamber was thawed for approximately 20 min. The term AFSCs in the mini chamber were aspirated using closed sterile technique without exposure to air with an18 gauge needle and a 20 mL syringe. A 100 µL sample was collected into an Eppendorf tube. 10 mL of normal saline was used to dilute up the cells in the syringe and another 100 µL aliquot was taken using closed sterile technique and with time point 0 starting now. The sample, combined with the 10 mL normal saline wash, was then allowed to sit in the syringe for 3 h and at each hour time point, the syringe was inverted five times before a 100 µL aliquot was taken and viability and count assessed. The diluted sample in the syringe was then passed through an Olympus EndoTherapy balloon catheter and collected in a 50 mL conical tube. Another 100 µL aliquot was collected into an Eppendorf tube and viability and count assessed. The catheter was washed with 15 mL of normal saline and collected in a 50 mL conical tube and 100 µL was taken and viability and count assessed. Finally all the AFSC that passed through the balloon catheter were combined for culture. The suspension was centrifuged and the supernatant was aspirated and the pelleted cells were resuspended in cell culture medium [4] and a 100 µL aliquot was taken. Viability and cell count for each aliquot was assessed as described. The cells were transferred into cell culture at 37 °C at 5% CO₂ and observed for cell growth.

Flow cytometry (fluorescence activated cell separation (FACS))

Cells were harvested and detached from a confluent plate and resuspended in an assay buffer with 5% fetal bovine serum (FBS) in 1X Dulbecco’s phosphate-buffered saline (1X DPBS). Cells were stained with 1:1,000 dilutions of antibodies to the surface markers in an assay buffer at 4 °C for at least 1 h and washed, centrifuged at 1,200 rpm for 5 min to pellet cells. The supernatant was decanted, the tubes taken to the Cytometry and Sorting Core facility at NYU Langone Health using a UVII Becton Dickinson Analyzer to run samples. Analysis of files via WinList program was performed to determine cell surface marker expression.

Spheroid production for differentiation assays

Plates of 0.8% of low melting agarose in alpha-MEM-GlutaMAX were prepared and filled the wells of tissue culture quality, round bottom 96-well plates with 100 uL of melted agarose. Term AFSC were seeded at a concentration of 10,000 cells in 100 uL to each coated well. The plate was incubated for two to three days for the three dimensional cell cultures to form [5].

Neural differentiation

Transferred 1 mL cell culture base medium into two wells of a 4-well chamber slide each and 1 mL of STEMDiff Neural Differentiation Medium into the remaining two wells. Transferred five spheroids into each well and incubated at 37 °C for 3 weeks with media changes every two to three days. Aspirated off media and washed wells with 2 mL 1X DPBS three times. Fixed spheroids with 3.7% buffered formalin at room temperature overnight. Washed with 2 mL 5% heat inactivated horse serum in 1X DPBS three times. Permeabilized spheroids/cells with 1% Triton X-100 5% heat inactivated horse serum in 1X DPBS overnight at room temperature. Washed with 5% heat inactivated horse serum in 1X DPBS at room temperature. Incubated 1:100 dilution AlexaFluor488-mouse-anti-human beta tubulin III and AlexaFluor647-mouse-anti-human Nestin in 5% heat inactivated horse serum in 1X DPBS at room temperature overnight. Washed with 2 mL 0.1% Tween-20 in 1X DPBS three times followed by three washes with 1X DPBS. Mounted slides with coverslips with VECTASHIELD Mounting Medium with DAPI and cured overnight. The cells were visualized under fluorescent microscopy with a Nikon Eclipse TE2000-E inverted microscope. The images were acquired with Nikon NIS-Elements Imaging Software BR 3.10. Images were analyzed and quantified using ImageJ 1.47v.

Chondrocyte differentiation

Transferred 1 mL cell culture base medium into two wells of a 4-well chamber slide each and 1 mL of differentiation medium into the remaining two wells. Transferred five spheroids into each well and incubated at 37 °C for 3 weeks with media changes and processing as above. Incubated 1:100 dilution rabbit-anti-human Aggrecan in 5% heat inactivated horse serum in DPBS at room temperature overnight. Washed with 2 mL 0.1% Tween-20 in DPBS three times followed by three washes with 1X DPBS. Incubated 1:200 dilution of the secondary antibody AlexaFluro594 goat anti-Rabbit IgG (H + L) in 5% inactivated horse serum in DPBS at room temperature overnight. Washed three times with 0.1% Tween 20 followed by three washes with 1X DPBS. Slides were prepared and analyzed as above.

Pancreatic differentiation

Transferred 1 mL cell culture base medium into two wells of a 4-well chamber slide each and 1 mL of differentiation medium into the remaining two wells and spheroids prepared as above. Incubated 1:100 dilution PE-mouse-anti human PDX1 and AlexaFluor488-mouse anti-human Insulin in 5% heat inactivated horse serum in 1X DPBS at room temperature overnight. Processing was described above for analysis.

Limitations

This study used a hemocytometer and trypan blue for manual cell count, and three to eight fields of visions were assessed. Publications support the use of at least three fields of vision when using this method of cell counting [6]. In comparison to other methods such as automated cell counting devices like the Vi-CELL^® XR Cell Viability Analyzer (Beckman Coulter) and manual hemocytometer displayed no significant differences in cell counting capabilities [7]. However, using a hemocytometer requires a level of skill on the part of the user to be reliable [8]. Practical considerations such as transport delays after collection to the processing facility might affect the viability of term AFSC.