Transwell system

Normal human skin biopsies were obtained as resected material after cosmetic surgery procedures. Four millimeter biopsies (n = 3) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, 1% pyruvate, 1% HEPES, and 2% penicillin/streptomycin and stimulated with 100 nM calcipotriol, DMSO, or a cytokine mixture (see above). A calcipotriol concentration curve was performed at 10, 30, 100, and 300 nM (n = 1). To study the effects of a topically applied commercially available VD3 ointment (calcitriol; Silkis®, Galderma, Switzerland), calcitriol ointment or petrolatum were applied to a 20 cm² piece of full thickness (including subcutis) normal human skin (n = 3), for 30 min at 37 °C. One third of the skin surface was left untreated and was used as control. After incubation, 4 mm biopsies were taken and cultured at air-liquid interface in the medium described above on a trans-well system (Greiner Bio-One, Germany) for 24 h, 48 h, and 72 h. Culture media level was adjusted to ensure optimal air exposure of the epidermis.

Spheroid formation of mono- and co-spheroids was performed using agarose molds, which were cast in MicroTissues^®3D Petri Dishes^® (16 × 16 arrays, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions (Figure 1). Here, 2.56 × 10⁵ cells were seeded per agarose mold (one agarose mold per well in a 12-well plate) and cultured in growth medium containing 10% FBS and 1% penicillin/streptomycin for two days, resulting in 256 multicellular spheroids per agarose mold (1000 cells/spheroid). Monocultures consisted of 1000 cells of the respective cell types, while co-cultures contained 500 ASCs and 500 cells of either MDA-MB-231 or MCF-7. For direct 2D co-cultures, 1 × 10⁴ cells/cm² of ASCs and breast cancer cells were seeded in a 6-well plate. For indirect 2D co-culture, a transwell system (Greiner Bio-One, Frickenhausen, Germany) was used, in which 1 × 10⁴ ASCs/cm² were seeded in the lower chamber and the same amount of breast cancer cells in the upper chamber. Accompanying all co-cultures, respective monoculture controls were conducted. Cells were cultured for 48 h before harvest. For imaging, ASCs and tumor cells were pre-stained with PKH26 (for tumor cells) and PKH67 (for ASCs, both Sigma-Aldrich) prior to spheroid culture according to the manufacturer’s instructions. Whole mount samples were imaged using an LSM780 confocal microscope (Zeiss, Jena, Germany).

A transwell system equipped with chambers (0.4 μm pore size; Greiner bio-one, Frickenhausen, Germany) was used to study the intercellular communication between microglia and primary neurons or astrocytes. This design facilitated the exchange of secreted substances between cells without requiring direct cell-to-cell contact [27 (link)]. Primary neurons or astrocytes were seeded on 24-well plates, respectively. Simultaneously, neurons underwent 10 h exposure to OGD, while astrocytes experienced 8 h of OGD [25 (link), 28 (link)]. Concurrently, primary microglia were seeded onto a 24-well insert chamber. To illustrate whether knockdown of NEAT1 in primary microglia affects neurons and astrocytes in our oxygen-glucose-deprivation/reoxygenation (OGD/R) models, microglia were treated under four different treatment conditions (normoxia, OGD, OGD + ASO scramble, OGD + ASO NEAT1). Thereafter, microglia were then introduced into the 24-well plates containing hypoxia-treated neurons and astrocytes followed by co-cultured under normoxic conditions for 24 h.