Beriplast p combi set

A total of 5 × 10⁴ rat amnion-derived MSCs were suspended in 50 μl of fibrinogen solution (from Beriplast P Combi-set, CSL Behring). This suspension was then placed in a single well of a 24-well plate and 50 μl of thrombin was then rapidly added to the well and mixed using the pipette tip. This formed the FG-MSC complex, which added with 100 μl of growth medium (αMEM with 20% inactivated FBS containing l-glutamine, penicillin and streptomycin). For the Control group, 5 × 10⁴ rat amnion-derived MSCs were plated on a 24-well plate with 200 μl growth medium. These were incubated at 37 °C and 5% CO₂. At specific time points, cells were dissociated by adding 500 fibrin-degradation units of nattokinase dissolved in 100 μl of αMEM. This detached the MSCs from the well. Viability and number of collected MSCs were assessed using Countess Cell Counter (Invitrogen).

Lobar resection is a lobectomy (removal of a single lobe) or bilobectomy (removal of 2 adjacent lobes). Sublobar resection is wedge resection or segmentectomy. We routinely used the PGA sheet (Neoveil large size; 10.0 x 10.0 cm: Gunze, Kyoto, Japan) and 3 ml or 5 ml of fibrin glue (Bolheal: Teijin, Tokyo, Japan or Beriplast P Combi-Set: CSL Behring, Tokyo, Japan) to cover the dissected hilum when we performed a lobectomy, a bilobectomy and a segmentectomy, regardless of a lung parenchymal air leak. In the case of wedge resection, we covered them to the point of the air leak only when we confirmed the presence of an air leak.

Fetal intervention of RA-exposed Sprague-Dawley rats was performed at E20. Pregnant rats were anesthetized by isoflurane, and an abdominal midline incision was made to expose the uterine horns. The MMC defect was confirmed through the uterus under direct vision, and a small incision of the uterine wall and amniotic membranes was made directly above the defected site. 3D skin was transplanted into the area of defected skin. Beriplast P Combi-Set (CSL Behring, #87799) was used for tissue adhesion. The hysterectomy site was closed by purse-string suture with 6-0 silk (Natsume Seisakusho, #B10-60). The uterus was returned to the abdomen and the abdominal incision site was closed by running suture. The fetuses were harvested by cesarean section at E22. Pregnant rats were then euthanized by cervical dislocation under anesthesia with isoflurane. The transplantation site was dissected and processed for histological and immunohistochemistry analysis. The primary antibody list is provided in Table S2. Appropriate Alexia 488- or Alexa 594-conjugated secondary antibody was used with DAPI nuclear counterstain. The operation protocols were accepted by the Laboratory Animal Care and the Use Committee (A2015-003-C01).