Cellbind surface

The biological activity of the VEGF released from the microcapsules was tested in the HUVEC (human umbilical vein endothelial cells) (PromoCell GmbH, Germany) cells because VEGF induces high proliferation in these cells^{61 (link)}. HUVEC cells were maintained in endothelial cell growth medium (PromoCell GmbH, Germany) supplemented with a supplement kit and were cultured in special cell culture flasks (Corning CellBIND Surface) at 37 °C in humidified air with 5% CO₂. After 2 or 3 passages, cells were seeded into 96-well poly-L-lysine-coated culture plates at a density of 5·10³ cells/well. After 24 h, the culture medium was removed and fresh media (without the supplement kit) was added and supplemented with one of these options: 0.8 ng/ml of VEGF released from BHK-VEGF containing microcapsules, 1 ng/ml of VEGF commercial solution as a positive control, or the released medium from empty microcapsules as a negative control. Cells were incubated for 72 h and cell proliferation was measured by the Cell Counting Kit-8 (CCK-8 assay) as explained previously.

For all experiments, human adipogenic tissue-derived mesenchymal stem cells (MSCs) were used. After obtaining the donor’s informed written consent, as approved by the Institutional Review Board (Hannover Medical School) with the reference number 3475-2017, adipose tissue was received following abdominoplasty surgery. After isolation, MSCs have been extensively characterized by surface marker analysis and functional properties as described earlier [26 (link)]. Cultivation of MSCs was performed in cell culture medium in a 5% CO₂, 21% O₂, humidified atmosphere at 37 °C (Heracell 150i incubator, Thermo Fisher Scientific Inc., Waltham, USA). The MSCs were routinely maintained in 75 cm² cell culture flasks (Corning, CellBind Surface, Corning, NY, USA), and then harvested at about 85% confluency by accutase treatment (Merck KGaA, Darmstadt, Germany) for detachment [26 (link)]. 24 h prior to the start of an experiment, cells were seeded in 6-, and 96-well plates (at a density of 18,000 cells·cm⁻² and 1100 cells·cm⁻², respectively) (Sarstedt AG and Co. KG, Nürnbrecht, Germany). Experiments were performed with cells of passages two to six.

Tissue fragments were placed in 115 cm² tissue-culture flasks with reclosable lids (TPP, Switzerland) in the presence of StemMACS-MSC expansion Media kit XF (Miltenyi Biotec GmbH, Germany) after coating with CELLstart^TM CTS^TM (Gibco/Thermo Fisher Scientific) or Synthemax^® II-SC (Corning) as adhesion substrates for “GMP grade I” and “GMP grade II” conditions, respectively (see Table 1). For the coating with CELLstart^TM CTS^TM, flasks were incubated with the reagent diluted 1:50 in DPBS for 2 h at 37°C. Regarding the coating with Synthemax^® II-SC, flasks were incubated with the reagent diluted 1:40 in sterile water for 2 h at RT.
At confluence, CPC were harvested using TrypLE^TM Select Enzyme (Gibco/Thermo Fisher Scientific), then seeded at 8–10 × 10⁴ cells/cm² and expanded in T flasks (75–150 cm²), HYPERFlask^® (1720 cm²) or HYPERStack^®-12 (6000 cm²) culture vessels (Corning). Thanks to their negatively charged, highly hydrophilic CellBIND^® surface (Corning), designed to facilitate cell attachment and spreading, HYPERFlasks and HYPERStacks did not require any coating.
For CPC MCB and PPCB generation (see below), the above indicated StemMACS-MSC expansion Media kit XF was substituted by its GMP counterpart, MSC-Brew GMP Medium (test lot kindly provided by Miltenyi Biotec GmbH).
CPC expanded in GMP grade conditions I and II are thereafter indicated as CPC-I and CPC-II, respectively.

HT-29 cells were propagated in Sarstedt culture flasks (25 cm²) in a 5% CO₂ humidified atmosphere at 37°C. Cells were fed with DMEM (Lonza) supplemented with 10% heat-inactivated FBS (Biosera) and 2 mM L-glutamine (Lonza). For the intracellular calcium measurement assay, cells were seeded in 24-well microplates (Corning CellBind Surface) at a density of 10⁵ cells/well. After 16 hr, the culture medium was replaced with fresh medium containing 5 μM of the calcium indicator Fluo-8 AM (AAT Bioquest), 4 mM of Probenecid (Sigma-Aldrich), and 0.025% (w/v) Pluronic acid F-127 (Invitrogen) according to a method adapted from Abrahamse and Rechkemmer, 2001 (link), and cells were incubated for 30 min at 37°C, 5% CO₂. The medium was removed, and calcium-free PBS was added before the cells were incubated at 37°C for another 30 min. The cell monolayer was washed twice with calcium-free PBS and PBS was added. Fluo-8 fluorescence was measured immediately after addition of Ionomycin (Sigma-Aldrich) 1 μg/ml, PBS, or purified OMVs (corresponding to 10 μg of soluble protein) with a FLUOstar Optima fluorescence plate reader (BMG Labtechnologies) fitted with custom excitation/emission filters (485/538 nm). OMVs were purified from 500 ml culture supernatants, washed with calcium-free PBS, and concentrated to a 2 ml suspension.

In order to minimize the presence of non-specific cells in the cellular product, the sample was enriched for CTLs by a plastic adherence process [16 (link)]. In brief, 2.25 × 10⁸ cells were plated in sterile 225 cm² A/N flasks with CellBIND Surface (Corning, Corning, NY) at 0.5 × 10⁶ cells/ml in X-VIVO 15 Serum-free cell medium w/o supplements (Lonza). Cells were incubated for 1 h at 37 °C and 5 % CO₂. Non-adherent cells were carefully collected by aspiration to avoid the disruption of adherent myeloid cellular populations and were washed with Dulbecco’s phosphate buffered saline (dPBS; Sigma-Aldrich) before quantification.

Permission to collect and use HPMCs was approved by the Institutional Human Subjects Review Board of the University of Texas Health Science Center at Tyler [Protocol code, 2020-039 (Date of approval, 14 September 2020)]. All experiments regarding human subjects were performed in accordance with relevant guidelines and regulations. Cells were isolated from pleural fluids collected from patients with congestive heart failure or post coronary bypass pleural effusions [61 (link)] and maintained on dishes with CellBIND surface (Corning, New York, NY, USA) using LHC-8 culture medium (Thermo Fisher Scientific, Waltham, MA, USA) or BEGM (without retinoic acid and epinephrine, Lonza, Basel, Switzerland) containing 3% fetal bovine serum (Thermo Fisher Scientific), 2% antibiotic-antimycotic (Thermo Fisher Scientific), and 1% L-glutamine (Thermo Fisher Scientific) in a humidified incubator at 37 °C and 5% CO₂/95% air.

Mouse macrophage cells RAW 264.7 and human gastric adenocarcinoma AGS cells were provided by Korean Cell Line Bank (Seoul, Korea). RAW 264.7 cells were cultured in DMEM (WelGene, Gyeongsan, Gyeongsangbuk-do, Korea) with 10% fetal bovine serum (FBS; WelGene) at 37 °C with 5% CO₂. AGS cells were grown in RPMI 1640 medium containing 25 mM HEPES supplemented with 10%, 50 units/mL penicillin, and 50 μg/mL streptomycin (WelGene) at 37 °C with 5% CO₂.
RAW 264.7 and AGS cells were seeded into 24-well tissue culture plates (Corning CellBIND Surface, Corning, NY, USA) at a density of 5 × 10⁴ cells/mL and incubated at 37 °C in a 5% CO₂ atmosphere. The cells were treated with 31.25, 62.5, 125, 250, 500, and 1000 μg/mL of CLCP fractions. After incubating for 24 h at 37 °C with 5% CO₂, RAW 264.7 cells were then stimulated with LPS (1.0 μg/mL) for 6 h, supernatants were collected and the cells were washed three times with PBS. Then, 10 μL of MTT solution (5 mg/mL) was added to each well. After incubating for 4 h, the supernatant from the wells was carefully discarded, and 100 μL dimethyl sulfoxide was added. The optical density was evaluated at 490 nm by the multi-plate reader in three replicates.