Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

The Human Research Ethics Committees at Mater Health Services, Royal Brisbane and Women's Hospital, Queensland University of Technology and the University of Queensland approved the research and collection of human placenta samples used in the study. All protocols complied with national research guidelines. Patients provided informed written consent for the use of tissue for research purposes.

Third trimester placentas were obtained from healthy mothers following routine Caesarean section (CS) births at term from the above-mentioned hospitals in Brisbane, Australia. Male discordant pregnancies for term samples were utilized in this study to distinguish fetal from maternal cells. Fetal gender had been determined by ultrasound prior to birth and/or visual inspection of the neonate at birth by clinical staff. X and Y chromosome fluorescence in situ hybridization (FISH) was utilized to further validate gender and fetal or maternal origin of tissue preserved from the original placentas.

1. Prior to Harvest, Prepare the Following

Note: When making up enzyme solutions, the specific requirements and concentrations provided by the manufacturer for each product should be followed. Enzymes are often provided as a crude mix of proteins, therefore similar enzymes from different companies are likely to have different activities/concentrations. For this reason the manufacturer's advice should be acknowledged and solution preparation may have to be modified accordingly.

1. Prepare or purchase 1.5 to 2 L total sterile Hank's Balanced Salt Solution (HBSS) for this protocol.
2. Prepare collagenase I stock solution by weighing out collagenase type I and dissolve 1 mg/ml in HBSS and vortex gently to ensure complete dissolution. Determine the volume of HBSS (containing calcium and magnesium) required to bring collagenase solution to 100 units (U)/µl (1,000x stock solution) and then filter using a 0.2 µm syringe filter. Aliquot 1 ml volumes into 1.5 ml tubes and store at -20 °C until needed.
3. Prepare dispase stock solution by dissolving non-sterile dispase at 10 mg/ml in Dulbecco's phosphate-buffered saline (DPBS) without calcium or magnesium. Further dilute with DPBS without calcium or magnesium to a final concentration of 2.4 U/ml. Filter sterilize through a 0.2 µm syringe filter. Aliquot 1 ml volumes in 1.5 ml tubes and store at -20 °C until needed.
4. Prepare deoxyribonuclease (DNase)-I stock solution by dissolving DNase-I at 10 mg/ml in 0.15 M NaCl. Filter thorugh a 0.2 µm syringe filter. Aliquot 1 ml volumes in 1.5 ml tubes and store at -20 °C until needed.
5. Prepare the working digestion solution by combining 100 U/ml collagenase type I, I.5 µg/ml DNase I and 2.4 U/ml dispase in serum-free DMEM. Freshly defrost and dilute enzyme stocks immediately prior to use. Approximately, a 1:1 ratio of digestion solution volume to tissue volume is required (e.g.10 ml of tissue, measured in a 50 ml tube, requires 10 ml of digestion solution).
6. Prepare a solution of 4% paraformaldehyde (PFA) in PBS and adjust to pH 7.4 ¹⁵. Store 25 mL aliquots at -20 °C for long term or at 4 °C for 1 week.
7. Prepare MSC culture medium from DMEM-Low Glucose (DMEM-LG) supplemented with 1x antibiotic and antimycotic solution and 10% non-heat inactivated fetal bovine serum. MSC-grade FBS can be purchased from many FBS suppliers.
8. Autoclave sterilize dissection equipment in advance as per institutional guidelines. These include scissors, forceps, scalpels, and a large metal dissection tray. Collect additional sterile tissue culture plasticware and consumables (e.g. scalpel blades, 50 ml and 15 ml tubes, 25 ml, 10 ml and 5 ml pipettes, and 75 or 175 cm² cell culture flasks).
9. Use standard primary cell culture laboratory equipment: a class II biosafety cabinet suitable for primary cell isolation, cell culture incubator set at 5% CO₂ and 37 °C, a rocker in a 37 °C incubator, and a centrifuge with a capacity for 50 ml tubes.
10. Use appropriate personal protective equipment: a laboratory coat, 2 pairs of gloves (at all times), safety glasses, and close-toed shoes as per institutional guidelines.
11. Prepare appropriate decontamination solutions for human blood specimens as per institutional guidelines and liquid biological waste containers in advance.

2. Isolation of the Placental MSC

1. Preparation of the Placenta
   1. Dress in the required personal protective equipment. Set up the biological safety cabinet with required materials, solutions and waste containers to carry out the procedure up to the enzymatic digestion (section 2.5.6).
   2. Obtain a placenta with informed consent and ethical approval. Collect the placenta via cesarean section under aseptic surgical conditions, and package in a sterile bag or bucket for transport to the laboratory.
   3. During transport and prior to dissection, store the placenta at room temperature or 4 °C.
      Note: Tissue can be stored for up to 6 hr without affecting cell culture performance.
      1. Limit the time between placenta collection and tissue processing where possible. This recommendation is based on practical issues. In some instances, we have delayed the cell isolation process for up to 6 hr after birth. In these instances placentas have been kept at room temperature or 4 °C (in the lab or at the collection center). No detectable differences were observed, above general donor variation, in the MSC obtained from placentas when they were stored at 4 °C or at room temperature.
   4. Place the container with placenta in the biological safety cabinet. Open the container and transfer the placenta into a sterile tray.
   5. Open out the fetal membranes (amniotic sac or amnion-chorionic laeve) ⁸. Become orientated with the parts of the placenta. The fetal side has the umbilical cord insertion, which is in general, centrally inserted in the placenta. The maternal side (lined with decidua basalis), which is opposite to the fetal side, has obvious cotyledons (or lobes).
   6. To begin the dissection, orientate the placenta with the umbilical cord facing upwards in the sterile tray.
      Note: This protocol is designed to yield sufficient cells to seed into one T175 flask from each of the three tissue types. Each digest begins with approximately 10 g of tissue. This is a similar starting number of flasks as MSC cultures initiated from a 20 ml bone marrow aspirate. If more cells are desired, then more tissue can be harvested and the protocol can be scaled linearly. The sizes of tissue to be harvested are only indicative of what is practical with the placental tissue. There are few defining features in the placental villi, which can be used as a reference point. The indicated dimensions serve as an estimate and the umbilical cord is used as a landmark on the fetal surface.
2. Dissection of the Decidual (D) Tissue
   1. Flip the placenta over so the maternal surface (decidua basalis) is facing up. Ensure that the fetal side with the umbilical cord insertion point is facing down.
   2. Cut pieces of 0.5 cm thickness from the maternal side of the placenta (containing decidua basalis tissue).
   3. Place tissue pieces into a Petri dish containing HBSS during the dissection to keep the tissue pieces hydrated.
   4. Transfer sufficient tissue to fill a tube up to the 10 ml mark in a 50 ml tube (this is approximately 10 g of tissue).
3. Dissection of the Chorionic Plate (CP) Tissue
   1. Mechanically remove of the amniotic membrane from the fetal surface of the placenta (not the amniotic sac), leaving the chorionic frondosum (chorionic plate) intact.
   2. Cut pieces ~1 cm wide by 0.5 cm deep from the chorionic plate. Harvest the chorionic plate from the region nearest to the umbilical cord, away from the edge of the placenta.
   3. Place tissue pieces into a petri dish containing HBSS during the dissection to keep them hydrated.
   4. Transfer sufficient tissue to fill a 50 ml tube up to the 10 ml mark (~10 g of tissue).
4. Dissection of the Chorionic Villi (CV) Tissue
   1. Dissect CV tissue ~1 cm² x 0.5-1 cm deep from placental tissue where the CP has already been removed. Again, harvest CV from the region closest to the umbilical cord, away from the edge of the placenta. Try to stay at least 1 cm away from the maternal side of the placenta (containing decidua basalis tissue); the goal is to harvest tissue from the interior of the placental villi tissue.
   2. Place tissue pieces into a Petri dish containing HBSS during the dissection to keep them hydrated.
   3. Transfer sufficient tissue to fill a 50 ml tube up to the 10 ml mark (~10 g of tissue).
5. Mincing and Enzymatic Digestion of the D, CP, and CV Placental Tissues
   1. As there are ~10 g of D, CP or CV tissues in three different 50 ml tubes, fill each tube with approximately 40 ml of HBSS and repeatedly invert the tube in order to wash the tissue (repeat for ~10 sec).
   2. Decant the supernatant and repeat this wash step 2-3 times until the solution is largely free from blood. Use a 10 ml pipette or long tweezers to ensure the tissue pieces are not lost out of the tube(s) when decanting the supernatant.
   3. Return tissue pieces to a 10 cm petri dish with minimal liquid transfer. Chop/mince the tissue into fine pieces of approximately 1-5 mm³ with scissors or razor blade.
   4. Transfer the minced tissues back into appropriately labeled (D, CP or CV) 50 ml tubes.
   5. Add freshly prepared digest media in at least a 1:1 ratio with the tissue (e.g. 10 ml of tissue plus 10 ml of digest media). Replace the cap on each tube and invert the tubes several times to mix.
   6. Incubate the tissues in the tubes with digest media for 1-2 hours at 37 °C. A humidified atmosphere and 5% CO₂ are not necessary for this step.
      Note: Rapid shaking is required in order to efficiently dissociate cells from the tissue and maximize MSC yield. Limited yield will be evident by very few cells attached to the culture flask at 48 hr and a first passage time longer than 1-2 weeks. The incubation time and shaking method is dependent on the 37 °C incubator available in the laboratory and may require optimization.
      1. For an incubator with a rapid and controllable shaking mechanism, place tissue and digest medium in a 50 ml tube or sterile conical flask, place in incubator and set shaking speed to 250 rpm. This will result in a complete digest of the tissue in 1 hr.
      2. Alternatively, for an incubator with a gentle rocking or no rocking mechanism, manually shake the tube rapidly and vigorously by hand for 10 sec every 30 min for 1.5 to 2 hr. The enzyme incubation period is a suitable time to discard waste, clear the biosafety cabinet of unnecessary items, change laboratory coat if soiled and prepare for the next part of the procedure.
6. Collection of Mononuclear Cells and Plating into Flasks
   Note: From the previous step, there are three 50 ml tubes (D, CP or CV) with digesting tissue pieces. When the tissue digest solution evolves a cloudy appearance and white blood vessels are obvious in the tissue, the digestion is complete.
   1. Add 30 ml of MSC media containing FBS to each tube to inactivate the enzymes contained in the digestion solution.
   2. To separate the mononuclear cells from the large undigested debris, pulse centrifuge the 50 ml tube. Briefly, allow the centrifuge to reach 340 x g for 5 sec and then stop. This will force the large debris to pellet at the bottom of the tube, while leaving the mononuclear cells in liquid suspension.
      Note: The mononuclear cells will remain in the liquid phase if the tube is only briefly (pulsed) centrifuged. If centrifugation is extended, the mononuclear cells will also pellet at the bottom of the tube with the tissue pieces, resulting in undesirable cell loss.
   3. Collect the supernatant, containing the mononuclear cells, and transfer this into a new 50 ml tube with a pipette.
   4. Add 30 ml of media or HBSS to the remaining placental tissue debris and shake vigorously. This will bring remaining detached mononuclear cells into suspension and enable a second harvest of putative MSC from the tissue.
   5. Pulse centrifuge the tube a second time, and again transfer the supernatant to a new 50 ml tube. Repeat this step a third time to maximize the collection efficiency of mononuclear cells from each tissue source.
   6. Pool the supernatants from each of the individual tissue types into two 50 ml tubes. This will yield 6 tubes total (2 tubes of each D, CP and CV). Centrifuge each tube for 5 min at 340 x g. This step will pellet the mononuclear cells to the bottom of the tubes.
   7. Carefully decant or pipette off the supernatant into the waste. It is unnecessary to try to eliminate every drop of supernatant.
      Note: The cell pellet will be fragile due to a large number of red blood cells. Be careful not to accidentally discard the pellet if you are decanting directly from the 50 ml tube.
   8. After removing most of the supernatant, gently flick the tube several times with a finger to dislodge the cell pellet. Combine the pellets, so that there is a single tube for each D, CP or CV tissue and resuspend each in 35 ml of MSC media.
   9. Optional: Filter the mononuclear fraction through a 100 µm mesh cell strainer set up in a 50 ml tube to remove cell clumps or fibrous material.
      Note: Care is required as the filters can easily clog up and over-fill. Additionally, air bubbles can obstruct filters from draining. If this occurs, slowly lift the filter out of the tube to allow air to flow under the filter, wash filter through with cell culture media or exchange to a new filter. Excluding the filtering step does not appear to alter yield or quality of the subsequent MSC cultures.
      1. Optionally, a red blood cell (RBC) lysis or density gradient centrifugation can be carried out at this stage ¹⁵. However, our experience is that RBC do not interfere with MSC attachment or proliferation and therefore RBC lysis is not necessary. Mononuclear cells could be counted at this step if RBC removal was carried out ¹⁵. Although, the vast majority of cells at this stage will not be MSC, but be fetal origin trophoblasts, endothelial cells and hematopoietic cells, as well as maternal origin hematopoietic cells and decidual stromal cells.
   10. Transfer the cell suspension for each D, CP or CV tissue into a single T175 flask and culture in a humidified incubator at 37 °C and 5% CO_2.
7. Post Isolation-cell Expansion
   1. At 48 hr following the initial isolation, remove the medium from each of the flasks (D, CP and CV) and replace with 35 ml of fresh MSC media. The spent medium and detached cells can be discarded, as putative MSC are presumed to be attached to the tissue culture flask ¹⁶. Return flasks to the incubator for a further 24 hr.
   2. Following an additional 24 hr of culture, wash the flasks twice with DPBS (25 ml) to remove debris and RBCs. Swirl the liquid around the bottom of the flask to dislodge RBCs. Use a pipette to remove liquid and debris. Add 35 ml of MSC media to the flasks and return the flasks to the incubator.
   3. Replace media twice per week, and incubate cultures until the cell monolayers are 80- 90% confluent. This initial expansion takes 4-14 days, depending on the quality of the tissue, amount of starting material and efficiency and time of incubation with the digest solution.

3. Subculture of pMSCs

1. When cultures are 80-90% confluent, wash twice with 20 ml 1x HBSS or DPBS and discard washes. Add trypsin-substitute to each flask (i.e. use 5 ml for a T175 flask).
2. Incubate the flasks for 5 min at 37 °C to liberate MSC from the tissue culture surface. Tap the sides of the flask every ~2 min to facilitate detachment.
3. When cells are detached from the surface and in a single cell suspension, wash the cells from the flask with MSC media and collect the cells in tubes. The MSC media will dilute and deactivate the trypsin-substitute (use ~15 ml of MSC media per T175 flask).
4. Transfer the contents of each tissue culture flask to a 50 ml tube.
5. Centrifuge tubes at 340 x g for 5 min to pellet the cells.
6. Discard the supernatant and resuspend the cell pellets in 1 ml of MSC media.
7. Dilute 10 µl of cell suspension in 10 µl trypan blue. Count cells using a hemocytometer and calculate the total number of cells ¹⁵.
8. Transfer cells to new flasks at 1,150 cells/cm² in fresh MSC media and incubate in a tissue culture incubator. At this seeding density, seed 200,000 MSC into each new T175 flask in 35 ml of MSC media. Feed cells twice per week until 80-90% confluent. Seed subsequent passages at 1,150 cells/cm² or 200,000 MSC into each T175 flask.
9. When the Passage 1 (P1) or P2 cells are confluent, cryopreserve the majority of the cells for future use, and reseed only one T175 flask for further propagation and characterization. The propagated cells can be used at P3 or P4 for mesodermal differentiation assays and cell characterization via flow cytometry.
   Note: In early passages, there may be very few sparsely separated colonies, but the local cell density within each colony may be very high. This is not ideal, as the local dense packing of the cells will result in contact inhibition, delaying growth and reducing culture performance. We recommend when dense colonies are observed, the cells should be harvested and reseeded into a new flask. This redistribution process provides cells with room to proliferate, and overall culture performance will be improved.
10. To cryopreserve MSC, collect ~1 x 10⁶ cells in 1 ml of 90% FCS and 10% dimethylsulphoxide (DMSO) and freeze using standard protocols.
    Note: In the representative results section, there are descriptions of MSC characterization using flow cytometry, mesodermal differentiation and XY FISH analysis to determine the gender of the three different (D, CP and CV) expanded cell populations. In our study, placentas were from male babies; thus all fetal cells could be distinguished as male (Y chromosome) and all maternal cells could be distinguished as female (X chromosome only).