Picm0rg50

The tumor cells, M2 macrophages, and autologous CAR-T or ESO-T cells were collected and mixed at ratios indicated in figure legends. Mixed cells were centrifuged and resuspended in C10 medium at 1 × 10⁵ cells per μL medium. The cell slurry was adjusted to 5–10 μL per aggregate and was gently transferred onto a microporous membrane cell insert (EMD Millipore, Billerica, MA, USA, #PICM0RG50) using a 20 μL pipet to form a 3D human tumor/TAM/T-cell organoid. Prior to cell transfer, cell inserts were placed in a six-well plate immersed with 1 mL C10 medium. Two days later, the organoids were dissociated by P1000 pipet tip and disrupted through a 70-μm nylon strainer to generate single-cell suspensions for further analysis. In some experiments, 50 μg/mL of anti-human PD-L1 antibody (B7-H1; clone 29E.2A3, BioXCell, Lebanon, NH, USA) or Mouse IgG2b, κ control antibody was added to the organoid to study the effect of anti-PD-L1 antibody to TAM-mediated T-cell antitumor reactivity suppression.

Heart slices were cultured on organotypic membranes (PICM0RG50, Merck Millipore, Billerica, MA, USA) [20 (link)] in Petri dishes that contained 1 mL of Medium 199 supplemented with penicillin/streptomycin and insulin/transferrin/selenite to guarantee nutrient supply. The slices were immediately placed in a CO₂ incubator with a temperature of 37 °C and a gas content of 5% CO₂ and 21% oxygen. Oxygen supply was guaranteed due to the direct contact of the slices’ surfaces with the air. After 24 h, the culture medium was changed, and the culture time was terminated after 48 h.

HSCs were prepared from pups at postnatal day 4–6 (P4–6) according to previously published protocols [27 (link), 28 (link)]. After decapitation, brains of the pups were aseptically removed, the hippocampi were dissected and cut perpendicular to the longitudinal axis into 350 μm sections with a tissue chopper. Carefully selected intact hippocampal sections were transferred into petri dishes containing an ice-cold buffer solution (minimum essential medium (MEM) supplemented with 2 mM GlutaMAX™ at pH 7.3). Three sections were placed onto a humidified porous polyethylene (PTFE) membrane insert (Merck Millipore, PICM0RG50) and into a 6-well plate with 1.2 ml culture medium (20% heat-inactivated horse serum in 1x MEM complemented with GlutaMax™ (1 mM), ascorbic acid (0.00125%), insulin (1 μg/ml), CaCl₂ (1 mM), MgSO₄ (2 mM) and D-glucose (13 mM) adjusted to pH 7.3) per well. HSCs were kept at 37 °C in humidified CO₂-enriched atmosphere. The culture medium was completely changed three times per week.

The membrane sensor is based on the existing membrane inserts made of hydrophilic PTFE with a diameter of 24 mm and average pore size of 400 nm (PICM0RG50, Merck Millipore, Billerica, MA, USA). The three electrode membrane based electrochemical biosensor requires a working electrode, counter electrode and a reference electrode, see Figure 1a. The working and counter electrodes were made of Au, while the reference electrode was made of AgCl. The pattern of the electrodes satisfies the requirement of having a larger surface area of the counter electrode than the working electrode [12 ]. The pattern of the electrodes deposited on the membrane, with electrical contacts from the membrane to the sides of the grips, is seen in Figure 1a.

Organotypic entorhino-hippocampal tissue cultures were prepared at postnatal day 4–5 from mice of either sex as previously described [29 (link)]. The tissue cultures were transferred onto porous (0.4 µm pore size, hydrophilic PTFE) cell culture inserts with 30 mm diameter (Merck/Millipore, Darmstadt, Germany, Cat# PICM0RG50) for cultivation. The culturing medium consisted of 50% (v/v) minimum essential medium (MEM), 25% (v/v) basal medium eagle (BME), 25% (v/v) heat-inactivated normal horse serum (NHS), 2 mM GlutaMAX, 0.65% (w/v) glucose, 25 mM HEPES buffer solution, 0.1 mg/mL streptomycin, 100 U/mL penicillin and 0.15% (w/v) bicarbonate. The pH of the culturing medium was adjusted to 7.30 and tissue cultures were incubated for at least 18 days at 35 °C in a humidified atmosphere with 5% CO₂. The culturing medium was replaced thrice a week at 2- and 3-day intervals. After the lesion, the medium was not replaced for 3 days until further experimental assessment.

Hippocampal slices were prepared from P6-P9 C57BL/6N mice as described previously [44 (link)]. Briefly, the animal was deeply anesthetized with isoflurane, after which the animal was quickly decapitated, and the brain removed. The hippocampi were isolated and cut into 350 µm sections in an ice-cold dissection medium (250 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 2 mM NaHCO₃, 4 mM KCl, 5 mM MgCI₂, 1 mM CaCl₂, 10 mM D-glucose, and 248 mM sucrose). The slices were cultured on the membrane inserts (PICM0RG50; Merck, Ireland), placed on the culture medium (50% minimal essential medium, 21% Hank’s balanced salt solution, 15 mM NaHCO₃, 6.25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 10 mM D-glucose, 1 mM L-glutamine, 0.88 mM ascorbic acid, 1 mg/mL insulin, and 25% horse serum), and incubated at 35°C in 5% CO₂.
The cultured slices at DIV3 were transfected with AAV using a glass pipette (Narishige). The preparation of AAV (titers typically ~5 × 10⁹ genome copies/µl) has been described previously in detail [45 (link),46 (link)]. For sparse labeling, AAVs encoding Clover-CaMKIIα and mCherry-Rem2 were coinfected with a low amount of AAV encoding Cre with the ratio of 100:300:4. On DIV13 or 14, two-photon FLIM-FRET experiment was carried out.

Paired ovaries from mice at 6 dpp were collected, and one ovary served as control and the other was incubated with CDC42 activator II (1 IU, Cytoskeleton, USA) for 30 min on an insert (PICM0RG50, Millipore, USA) in six-well culture dishes. Ovaries from mice at 35 dpp were cut into 4 pieces before treatment. The culture media was DMEM/F12 (GIBCO, Life Technologies, USA) supplemented with ITS (1:100, Sigma, USA) and penicillin-streptomycin solution. After the treatment, paired ovaries from the same donor were randomly inserted under each kidney capsule of the same ovariectomized host. Mice were sacrificed at 14 days after transplantation to assess follicular development. For Western blot assay, ovaries were incubated with or without CDC42 activator II on an insert in six-well culture dishes in culture media described above for 30 min, respectively. Ovaries were transferred to drug-free medium and cultured for 12 h and then harvested to Western blot analyses.