単離およびヒト胎盤組織由来の間葉系幹/間質細胞の増殖

The overall goal of this procedure is to isolate and expand mesenchymal stem stromal cells derived from human placenta tissue. Hi, I'm Mike Doran. I'm a group leader at the Queensland University of Technology at the Translational Research Institute in Brisbane, Australia.

Hi, my name is Rebecca Pelekanos, and I'm a researcher at the University of Queensland Center for Clinical Research. Today, I'll be demonstrating the MSC isolation procedure. The placenta is normally discarded following birth.

Because the tissue is five to 700 grams in weight, it offers a uniquely large source of allogeneic mesenchymal stem cells. Generally, people who are new to placental MSC isolation will struggle because they are unfamiliar with the placenta anatomy and unfamiliar with how to process the tissue and unfamiliar with what type of cells, maternal or fetal, will populate their final MSC culture. To assist researchers, we discuss these important details in this video.

The first step in this procedure is to orient yourself with the placenta anatomy. The second step in this procedure is to manually dissect 10 grams of tissue from either the decidua, chorionic villi, or the chorionic plate, using scissors. The first two steps are summarized again here schematically.

In step three, the decidua, chorionic villi, or the chorionic plate tissues are further minced into fine pieces with scissors. In step four, cells are liberated from the small pieces of tissue via a one-to two-hour digestion in dispase and collagenase I.In step five, the cells are separated from the fibrous tissue by washing them through a cell strainer. The cells are then collected, resuspended in culture medium, and put into culture flasks.

Mesenchymal stromal cells, MSCs, will be selected based on their propensity for plastic adherence and capacity to survive and proliferate in the cell culture medium. Finally, in the results section, the expanded MSC can be characterized and stored for use in future experiments. The placenta is a temporary organ that allows the exchange of nutrients and waste products between the fetus and mother during pregnancy.

The placenta can easily be collected aseptically following cesarian section and the safe delivery of the baby. The placenta is predominately comprised of fetal-derived vasculature and membranes. However, it also contains some maternal cells.

The placenta is connected to the uterus by both maternal, uterine-derived decidual cells and fetal-derived, invading trophoblasts. It is not usually possible to discern by eye the precise interface between maternal and fetal tissues. However, the fetal side of the placenta is easily identified as the side where the umbilical cord is located.

When the baby is delivered, only a thin layer of decidual tissue remains connected to the placenta. Through physical orientation of the placenta, it is possible to locate that the three tissues that we will harvest today. The first tissue will be harvested is maternal decidua.

The green marker points to the decidual tissue that remains on the surface after the placenta is shed from the uterine wall. The second tissue that will harvested from the interior of the placenta is the fetal chorionic villi. This is indicated by the blue circles on the diagrams.

The third tissue to be harvested will be fetal chorionic plate. This tissue is identified by the red markers on the diagrams. Okay, let's get started.

Place the placenta in the biological safety cabinet. Open out the fetal membranes and orient yourself with the parts of the placenta. The fetal side with the umbilical cord insertion, the chorioamniotic laeve, or amniotic sac.

The fetal surface of the placenta has obvious large blood vessels in the chorionic plate, with overlying amniotic membrane, which, again, is easily mechanically separated. The maternal side is opposite to the fetal side, with the obvious cotyledons or lobes. Ensure the maternal surface is facing up.

Cut pieces of 0.5 cm thickness from the maternal side of the placenta or decidua. During the dissections, place tissue pieces into a petri dish containing Hank's Balanced Salt Solution, or HBSS, to keep the tissues wet. Agitate the tissues in the dishes with HBSS to remove some blood.

Transfer sufficient tissue to fill the tube up to the 10 milliliter mark of a 50 milliliter tube, approximately 10 grams. Note, more tissue can be isolated at this stage if more cells are required at the end of the procedure. Ensure the fetal surface is facing up.

Mechanically remove the amniotic membrane from the fetal surface of the placenta, leaving the chorionic plate intact. Cut the chorionic plate into strips approximately one half centimeter deep into the villus tissue beneath. Then, dissect chorionic villi tissue approximately one centimeter deep into the placenta from where the chorionic plate has been removed.

Place the tissues into HBSS and measure approximately 10 milliliters of the tissue as was done previously. Transfer tissue pieces, with minimal liquid transfer, to a 10 centimeter petri dish and chop into fine pieces approximately one cubic milliliter, with scissors. Transfer the minced tissue back into the 50 mil tube.

Repeat for all the remaining tissue samples. Add the freshly prepared digest media in at least a one-to-one ratio with the tissue. Invert the tube several times to mix.

Incubate the tissues in the tube with digest media for one to two hours, at 37 degrees in a shaking incubator. When the tissue digest solution has changed to a cloudy appearance, and white blood vessels were obvious in solution, add 15 milliliters of HBSS to dilute the enzymes in the digest media. Briefly pulse centrifuge the samples only until the centrifuge reaches 340 g for five seconds, and then stop.

This will force the large debris to collect at the bottom of the tube. Return samples to the biological safety cabinet. Place a cell strainer into a new 50 milliliter tube.

Pipette the supernatant containing the mononuclear cells into the strainer, catching the eluate in the new tube. Leave the placental tissue debris behind in the first tube. Add 15 milliliters of HBSS to the placental debris, shake vigorously to recover more cells, and pulse centrifuge the sample.

Again, pipette the supernatant into the tube with the cell strainer in the collection tube. Pouring the supernatant into the cell strainer may be more suitable, depending on the consistency of the sample. Wash the cell strainer with 5 milliliters of HBSS.

Discard the tube containing the placental tissue debris. Centrifuge the supernatant sample for five minutes at 340 g to pellet the cells. The red blood cells are visible as a layer at the bottom of the pellet, and the mononuclear cells, including the MSCs are the lighter layer on top of the red blood cells.

Return samples to the biological safety cabinet. Carefully decant the supernatant and discard. Flick the tube several times with a finger to dislodge the cell pellet, and resuspend the cell pellet in 35 milliliters of MSC media.

Transfer the cell suspension to one T175 flask per 10 mils of tissue, and culture in a 37 degree incubator with 5%CO2. After isolation, change the culture media after 48 hours. Feed the cultures once per week until confluent.

Cultures should be confluent by approximately 14 days. Finally, expand and cryopreserve cells, analyze MSC characteristics, and use in experiments. Following 48 hours of culture, MSC will have attached to the tissue culture plastic, while hematopoietic and other cell populations will not have.

At this time, the medium is replaced with 35 milliliters of fresh culture medium. The appearance of the culture supernatant can vary substantially between placenta donors. Example one and two demonstrate this variation.

These two isolations were performed simultaneously, but from two different placentas. Once washed, cultures are clear of red blood cells and tissue debris as shown in example three, and subsequent expansion results are generally consistent. After the 48 hour media change, only a few cells are attached to the flask.

Seven days after isolation, fibroblastic MSC colonies are visible, although non-MSC cells are also present, as round or loosely attached cells. 13 days after isolation, fibroblastic MSC colonies are large, and often the monolayer of MSC is sufficiently confluent and ready to passage. From passage to onwards, the MSC monolayer develops a characteristic whirlpool-like morphology at confluence.

The placental MSC display a classic MSC marker profile by flow cytometry analysis. The cells are positive for CD73, CD105, and CD90, and negative for the hematopoietic markers of CD45, CD34, and HLA-DR. The placental MSC generally undergo a robust osteogenic differentiation but poor adipogenic differentiation.

Many publications assume that cells isolated from the fetal chorion yield fetal MSC upon culture. However, as we have reported previously, all cultures derived from fetal chorion using this protocol rapidly become enriched for maternal MSC, as would be expected for the maternal decidual MSC cultures. The data shown here quantify the fetal and maternal cell composition of the placental MSC cultures isolated from decidual, chorionic villi, and chorionic plate tissues.

After watching this video, you should have a pretty good idea of how to isolate and culture MSC derived from placental tissue. We hope that you find these methods useful in your research and clinical applications that might use placental-derived MSCs.