Dynamic matrices with DNA-encoded viscoelasticity for cell and organoid tradition

Supplies

Solvents and reagents had been bought from business sources and used as obtained, except in any other case specified. Methanol (ACS reagent grade) was obtained from Fisher Scientific. Molecular biology grade acrylamide (catalogue quantity A9099), sodium acrylate (catalogue quantity 408220), 19:1 acrylamide/bis-acrylamide (catalogue quantity A2917) and ammonium persulfate (APS; catalogue quantity A3678) had been bought from Sigma-Aldrich. Ultrapure N,N,N′,N′-tetramethylethylenediamine (TEMED; catalogue quantity 15524010) and SYBR Gold Nucleic Acid Gel Stain (catalogue quantity S11494) had been obtained from Thermo Scientific. Desalted oligonucleotides had been bought from Built-in DNA Applied sciences (IDT). Nitrogen gasoline (>99.999%) was used underneath inert circumstances and provided by an in-house gasoline generator. To make sure an inert situation, it was purified by way of a Mannequin 1000 oxygen entice from Sigma-Aldrich (catalogue quantity Z290246). Reagents with unreacted acrylamide teams had been saved at 4 °C or −20 °C, shielded from pointless publicity to mild. Actin protein (>95% pure, rabbit skeletal muscle, catalogue quantity AKL95) was obtained from Cytoskeleton and DNase I (catalogue quantity M0303) was obtained from New England Biolabs. Matrigel was obtained from Corning (catalogue numbers 354277 and 356231). DNA ladders had been bought from Thermo Fischer (catalogue quantity SM1211).

Cell sources

Human bone marrow-derived MSCs had been remoted from wholesome feminine and male donors (aged 26–37) with knowledgeable consent by the Medical College on the College Hospital Dresden (Ethikkommission an der Technischen Universität Dresden, ethic board quantity EK263122004). MDCK II cells (ECACC 00062107) had been offered by A.H.’s group. Human pluripotent stem cells had been generated on the CRTD Stem Cell Engineering Facility, Technische Universität Dresden, by Shahryar Khattak and registered underneath the hPSCreg title CRTDi003-B (https://hpscreg.eu/cell-line/CRTDi003-B). The CT27 patient-derived trophoblast stem cell (TSC) line used on this examine was obtained from the RIKEN stem cell financial institution (https://cellbank.brc.riken.jp/cell_bank/CellInfo/?cellNo=RCB4936). The CT27 TSCs had been derived from placental cytotrophoblast cells as described in ref. ⁶². Human placentas had been obtained from wholesome ladies with signed knowledgeable consent of the donors, and the approval of the Ethics Committee of Tohoku College College of Drugs (Analysis license 2014-1-879).

Polymer synthesis

An in depth protocol is offered in ref. ⁴⁵. In short, acrylamide (50 mg ml⁻¹), sodium acrylate (0.5 mg ml⁻¹) and acrylamide-labelled anchor strand DNA (Supplementary Desk 1) had been co-polymerized in TBE buffer (100 mM Tris, 100 mM boric acid, 2 mM EDTA, pH 8.2). For P₁, P₅ and P₁₀, the DNA concentrations in resolution had been 100 µM, 500 µM and 1,000 µM, respectively. For the corresponding RGD-functionalized derivatives ({{bf{P}}}_{{bf{5}}}^{{bf{RGD}}}) and ({{bf{P}}}_{{bf{10}}}^{{bf{R}}{bf{G}}{bf{D}}}), 10 mM of acrylated RGD peptide (sequence, G(acryl-Ok)GGGRGDSP) was co-polymerized with the above resolution. Polymerization reactions for P₁, P₅ and ({{bf{P}}}_{{bf{5}}}^{{bf{RGD}}}) had been initiated with 0.005 wt% APS in presence of 0.005 wt% TEMED. Synthesis of P₁₀ and ({{bf{P}}}_{{bf{10}}}^{{bf{R}}{bf{G}}{bf{D}}}) was initiated by 0.025 wt% TEMED and 0.025 wt% APS. To realize excessive molecular weight and a slender dimension distribution, it was vital to hold out the response in high-purity nitrogen gasoline, which was handed by way of an oxygen entice on-site. The response was allowed to proceed in a single day, leading to a extremely viscous polymer resolution indicating the formation of lengthy polymer chains. NMR spectroscopy was used to confirm excessive conversion of the monomer (Supplementary Fig. 3). The answer was diluted in 9 volumes of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and subsequently purified by way of methanol precipitation. The pellet was resuspended in milliQ water and saved in aliquots at −20 °C. A small aliquot of the polymer resolution was lyophilized and weighted to find out the synthesis yield. Yields: 89% (P₁), 98% (P₅), 93% (P₁₀), 92% (({{bf{P}}}_{{bf{5}}}^{{bf{RGD}}})) and 88% (({{bf{P}}}_{{bf{10}}}^{{bf{R}}{bf{G}}{bf{D}}})).

Nearest-neighbour thermodynamic predictions

Express sequences of crosslinking strands and their enhances had been generated utilizing a customized Python script. This system sequentially replaces the ambiguous bases N with nucleobases (A, C, G, T) by way of multilayer nested loops. This created a library of ahead and reverse splint variants. The minimal free energies (MFEs) of all specific overlap domains had been calculated in opposition to all specific overlap domains within the library utilizing NUPACK (model 4.0.0.27), utilizing nearest-neighbour thermodynamic parameters based on ref. ⁶³. The mannequin parameters had been set to T = 20 °C, 150 mM NaCl, 75 µM complete DNA focus (37.5 µM for every splint strand), and a fancy dimension of two. For every potential pair, the Boltzmann distribution issue B_x,y was sequentially calculated for every variant of ahead (x) in opposition to all potential reverse splint (y) strand on the anticipated theoretical focus utilizing the equation

the place ok is the Boltzmann fixed. The normalized distribution was calculated to check the selective affinity of any ahead splint variant in a pool of reverse splint sequences. This was calculated for every pair because the ratio of distribution issue of 1 pair (x, y) by the sum of all pairs as

We observe {that a} exact resolution can solely be obtained by numerically fixing a system of odd differential equations⁶³. For simplification, we assumed that every splint strand is offered at its preliminary focus. In actuality, non-complementary splint strand concentrations are anticipated to be considerably diminished, as they’d be predominantly scavenged by their complementary binding companions. The obtained values for particular CCL pairs within the Boltzmann distribution which might be primarily based on our simplified calculation are due to this fact thought-about a decrease restrict for relative abundances.

Thermal annealing of DNA-crosslinked hydrogels

Until specified, the samples had been ready on the closing focus of 1% (w/v) polymer resolution, added with one equal of DNA crosslinkers to the anchor strand (for instance, 75 μM crosslinkers for 1% (w/v) P₅ resolution), within the buffer situation of 150 mM NaCl and 1x TE buffer. The samples had been annealed on the thermal cycler utilizing the next steps: (1) heating at 95 °C for 3 min, (2) instantaneous cooling from 95 °C to 80 °C, (3) holding at 80 °C for two min, (4) first cooling ramp from 80 °C to 65 °C at −0.3 °C min⁻¹, (5) second cooling ramp from 65 °C to 37 °C at −0.5 °C min⁻¹, (6) holding at 37 °C for twenty-four h.

The primary cooling ramp facilitates binding of the adaptor domains of the crosslinkers to the anchor strands, whereas the second ramp permits correct binding of the overlap domains to their complementary companions.

Oscillatory rheological measurements

Viscoelasticity measurements had been performed utilizing Anton Paar MCR301 rheometer with a 25-mm-diameter cone-plate geometry (cone angle 0.5°). Temperature management was ensured by putting in a Peltier system PTD-200 (Anton Paar) inside the measurement space. To keep up excessive humidity and forestall evaporation artefacts, a moist paper cylinder was positioned across the plate’s circumference. Amplitude sweeps had been carried out from 0.1% to 1,000% pressure at 1.6 Hz, whereas frequency sweeps had been performed from 0.1 Hz to 100 Hz at 10% pressure. Temperature sweeps lined a spread from 4 °C to 60 °C at 10% pressure, adopted by 60 °C to 70 °C at 20% pressure, all at 1.6 Hz. The heating and cooling steps had been carried out reversibly for 2 cycles to exclude evaporation artefacts. Until specified, samples had been measured in triplicate for reproducibility. Stress-relaxation properties had been measured at 15% pressure, the place the pressure was saved fixed whereas recording shear stress over time. Self-healing assessments concerned alternating between 1,000% and 10% pressure for five min every, repeated 4.5 occasions at a frequency of 1.6 Hz. For the heat-activated gelation assessments, two precursors had been ready at 1% (w/v) polymer resolution, added with: (1) 40 µM ahead CCL-4 strands (strand ID 4a) and 80 µM corresponding blocking strand (strand ID 14a), and (2) 40 µM reverse CCL-4 library (strand ID 4b) and 80 µM corresponding blocking strand (strand ID 14b). The precursors had been heated at 95 °C for 3 min, slowly annealed with a cooling ramp from 95 °C to 4 °C at −3 °C min⁻¹, and saved at 4 °C in a single day. Earlier than measurement, the 2 precursors had been blended and utilized onto the rheometer. The samples had been held at 4 °C for five min, shortly heated to 37 °C and maintained at 37 °C for 60 min with shearing at 10% pressure and a frequency of 1.6 Hz.

Hydrogel printing

A microscope glass slide (76 × 26 mm, Thermo Scientific) was connected to the printing mattress on BioScaffolder BS5.1 (GeSiM). Two-hundred microlitres of DyNAtrix (1% (w/v) P₅ + CCL-64) stained with SYBR Gold was transferred to a disposable 1 ml syringe (Omnican 40). To take away air bubbles, the syringe was centrifuged invertedly at 300g for two minutes. The hydrogels had been printed by way of a 30-gauge needle (Omnican) at room temperature, with a printing velocity of two mm s⁻¹, an extrusion charge of 40 μm s⁻¹ and a layer top of 0.12 mm. The ensuing 3D buildings, with a dimension of 20 × 20 × 2 mm (L × W × H), had been imaged on Opera Phenix confocal microscope (PerkinElmer) at ×5 magnification.

Mixing homogeneity assessments

Two precursor options had been ready in 1x TE buffer on the closing focus of 1% (w/v) P₅ added with: (1) 40 µM ahead CCL-64 strands (strand ID 6a) and 80 µM corresponding blocking strand (strand ID 15a), and (2) 40 µM reverse CCL-64 library (strand ID 6b) and 80 µM corresponding blocking strand (strand ID 15b). Ten micromolar of a 6-FAM (fluorescein)-modified strand (strand ID 15) was added to the precursor (1). After 1 h of equilibration at 4 °C, the 2 precursors had been blended utilizing a constructive displacement pipette. The combination was distributed right into a 384-well plate (Greiner Bio-One) at a quantity of 10 µl per nicely. The plate was then incubated at 37 °C for 30 min to provoke gelation. Lastly, photos had been acquired on Andor Dragonfly Confocal Microscope (Oxford Instrument) at ×4 magnification.

Enzymatic digestion assay

The Förster-resonance-energy-transfer-paired oligos, modified with Cy5 fluorophore and Iowa Black Darkish quencher (Q), had been bought from IDT. A mix of two µM Cy5-strand (strand ID 13a) and 4 µM Q-strand (strand ID 13b) was ready in 1x PBS buffer and subjected to annealing steps: heating at 95 °C for 1 min, instantaneous cooling to 50 °C for two min and gradual cooling from 50 °C to twenty °C at a charge of −1.5 °C min⁻¹. Samples had been ready with a closing focus of 100 nM Cy5-Q Förster-resonance-energy-transfer-probe and ranging actin content material from 2.5 µg ml⁻¹ to 320 µg ml⁻¹ (Cytoskeleton, catalogue quantity AKL95-B) in a buffer containing 150 mM NaCl, 1.8 mM CaCl₂, 0.2 mM ATP (Jena Bioscience, catalogue quantity NU-1010) and 1x IDTE. Earlier than measurement, 80 U ml⁻¹ DNase I (NEB, catalogue quantity M0303S) was added to every pattern. Every pattern was loaded in triplicate at a quantity of 20 µl per nicely right into a 96-well quantitative PCR plate. Fluorescence indicators had been recorded each 10 min for twenty-four h, and subsequently each 30 min for the next 36 h at 37 °C utilizing a Bio-Rad CFX96 Actual-Time PCR System.

Gel quantity and digestion measurements

The lyophilized actin protein (Cytoskeleton, catalogue quantity AKL95-B) was reconstituted with 100 µl deionized water to a focus of 10 mg ml⁻¹. The reconstituted actin was then in a buffer containing 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl₂, 0.2 mM ATP, 5% (w/v) sucrose and 1% (w/v) dextran. Citric acid powder (192.124 MW) was ready in 1x PBS to a closing focus of 200 mM, pH 7.0. The gel (1% (w/v) P₅ crosslinked with CCL-64) had been stained with 10X SYBR Gold earlier than swelling. The gel was then distributed right into a 384-well plate (Greiner, quantity 788092) at a quantity of 5 µl per nicely. The samples had been immersed in 20 µl of (1) 1x TE buffer, (2) 10% FBS-containing DMEM, (3) 50 µg ml⁻¹ actin + 10% FBS-containing DMEM, or (4) 10 mM citrate + 10% FBS-containing DMEM. Photos had been captured each hour for 48 h at 37 °C on Opera Phenix Plus confocal microscope (PerkinElmer) at ×5 magnification. The gel quantity was quantified utilizing IMARIS software program (Oxford Instrument) by way of the floor wizard and statistical instrument underneath a uniform threshold setting.

Entire blood incubation

Entire blood incubation was carried out as described beforehand^64,65. Gels had been ready on glass carriers with a polyethylene-alt-maleic anhydride bonding layer⁶⁶, and inserted in in-house developed incubation chambers fabricated from polytetrafluoroethylene, the place a 3.2 cm² check floor was uncovered to 2 ml blood⁶⁴. Reactively cleaned glass⁶⁷ and Teflon AF (DuPont) served as activating and inert reference surfaces, respectively.

Blood was obtained from two voluntary ABO-matched donors who had not used any drugs up to now ten days. The blood was instantly anticoagulated with 1.0 U ml⁻¹ heparin (Ratiopharm), pooled and stuffed into the incubation chambers with the samples, avoiding an air interface. The chambers had been incubated for two h at 37 °C at fixed overhead rotation to stop sedimentation. The blood was subsequently analysed for the blood cell rely (Beckmann Coulter AcTdiff). Granulocyte and monocyte activation had been decided by circulate cytometry (LSR Fortessa, Becton Dickinson), the place granulocytes and monocytes had been recognized for his or her scatter traits and CD15 (clone SSEA-1, PE-conjugated, BioLegend) and CD14 (clone M5E2, APC conjugated, Becton Dickinson) positivity, respectively. The depth of CD11b expression (clone ICRF44, PacificBlue-conjugated, BioLegend) was normalized to blood incubated with 100 EU ml⁻¹ endotoxin. The speed of granulocytes with conformationally activated CD11b was decided utilizing anti-CD11b clone CBRM1/5 (PE/Cy7-conjugated, BioLegend).

Soluble markers of coagulation activation (prothrombin fragment F1+2), blood platelet activation (platelet issue 4 (PF4)) and complement activation (complement fragment C5a) had been decided from plasma utilizing business enzyme-linked immunosorbent assays (ELISAs ; Enzygnost F1+2, Siemens Healthineers; Zymutest PF4, Hyphen BioMed; C5a ELISA, DRG Devices) after stabilization of the blood with the really useful components of the check kits and centrifugation.

The evaluation was carried out with a triplicate set of samples in parallel (n = 3). The examine was lined by the ethic vote EK-BR-24/18-1 of the Sächsische Landesärztekammer.

3D cell tradition

An in depth protocol is offered in Supplementary Strategies. In short, gel parts had been dissolved in water and saved at −20 °C for cell tradition. For MSC and hiPSC cultures, two precursors had been ready at a closing focus of 1% (w/v) ({{bf{P}}}_{{bf{5}}}^{{bf{RGD}}}) with: (1) 37.5 µM ahead splint (strand ID 6a) and 75 µM blocking strand (strand ID 15a), and (2) 37.5 µM reverse splint strands (strand ID 6b) and 75 µM blocking strand (strand ID 15b). As for MDCKII and hTSC cultures, two precursors had been ready at a closing focus of 1% (w/v) ({{bf{P}}}_{{bf{10}}}^{{bf{RGD}}}) with: (1) 75 µM ahead splint (strand ID 6a) and 150 µM blocking strand (strand ID 15a), and (2) 75 µM reverse splint strands (strand ID 6b) and 150 µM blocking strand (strand ID 15b). Concentrated DMEM was added to achieve a closing 1x focus. The precursors had been equilibrated at 4 °C in a single day for the blocking strands to bind quantitatively to the splint strands.

The cell suspension was blended with the primary precursor (1) after which with the second precursor (2). Cell-laden hydrogels (5 µl per nicely) had been positioned in 384-well plates or micro-well inserts (ibidi, catalogue quantity 80409) connected in 6-well plates. The samples had been incubated at 37 °C for 30 min to set off heat-activated gelation. After gelation, particular person tradition medium was added to every nicely. When utilizing serum-containing medium, 100 µg ml⁻¹ of actin (Cytoskeleton, catalogue quantity AKL95) was included to suppress nuclease exercise. The embedded cells had been cultured at 37 °C, 5% CO₂.

For Matrigel teams, cells had been embedded in 50% Matrigel. The cell seeding density, gelation time and medium had been similar to the DyNAtrix samples.

Fluorescence stay/useless assay and cell launch

MSCs had been stained with 3 µM calcein AM (PromoKine, catalogue quantity PK-CA707-80011) and 0.75 µM Draq7 (Invitrogen, catalogue quantity D15106) in medium for 30 min at 37 °C, 5% CO₂. Confocal photos had been taken on Opera Phenix Plus confocal microscope (PerkinElmer) at ×10 magnification. Cell counting was carried out with IMARIS software program (Oxford Instrument) utilizing the spots wizard. Cell viability was decided by dividing the variety of stay cells by the sum of stay and useless cells.

For cell launch, 2 U per nicely DNase I used to be added after the MSCs had been stained with 3 µM calcein AM for 30 min. Confocal photos had been acquired each 20 min at ×5 magnification for two h at 37 °C with 5% CO₂.

Immunofluorescent staining

hiPSCs had been mounted with 2% paraformaldehyde for 40 min at room temperature, adopted by permeabilization and blocking with 0.1% Triton X-100 in 2% BSA/PBS for 1 h °C. The cells had been then incubated in a single day at 4 °C with the first antibodies (1:300 anti-Oct3/4 (BD Biosciences, catalogue quantity 611202)) in 2% BSA/PBS. After washing the samples 3 times with PBS, secondary antibodies (1:200 Alexa Fluor 488, 1:200 Phalloidin ATTO 550 and 1:1,000 Hoechst 33342) in 2% BSA/PBS had been added and incubated in a single day at 4 °C. Confocal photos had been acquired utilizing Opera Phenix Plus confocal microscope (PerkinElmer).

As for hTSCs and trophoblast organoids, the samples had been mounted with 2% paraformaldehyde for 40 min at 4 °C, permeabilized with 0.5% Tween-20/PBS for 30 min and blocked with 3% BSA (Sigma) + 0.1% Tween-20 for 1 h. The cells had been incubated with the first antibodies within the blocking resolution for two days at 4 °C. The first antibodies used had been: anti-E-cadherin (Invitrogen, catalogue quantity 13-1900, 1:200), anti-Syndecan (Sigma, catalogue quantity HPA006185, 1:200), anti-GATA3 (R&D, catalogue quantity AF2605, 1:200), anti-ENDOU (Sigma, catalogue quantity HPA012388, 1:200), anti-GCM1 (Atlas, catalogue quantity HPA011343, 1:200) and anti-TEAD4 (abcam, catalogue quantity ab58310, 1:200). After washing with PBS 3 occasions, the cells had been then incubated with the secondary antibodies within the blocking resolution for two days at 4 °C. The secondary antibodies used had been: Alexa Fluor 488-, 594- and 647-conjugated antibodies. Nuclei had been stained with Hoechst (1:200). Confocal photos had been acquired utilizing Zeiss LSM 880 Ethereal inverted microscope and the ×63/1.3 LCI Plan-Neofluar goal with water immersion medium.

Ethics assertion

Knowledgeable consent was obtained from all recipients and/or donors of cells or tissues. The examine involving human complete blood was lined by the ethic vote EK-BR-24/18-1 of the Sächsische Landesärztekammer. The blood was obtained from two voluntary ABO-matched donors who had not used any drugs up to now ten days. The examine involving human bone marrow-derived MSCs was lined by the ethic vote EK221102004 and EK47022007 at TU Dresden. MSCs had been remoted from wholesome feminine and male donors (aged 26–37) by the Medical College on the College Hospital Dresden. The examine involving hiPSCs was lined by the ethic vote EK363112012 at TU Dresden. hiPSCs had been generated from MACS-sorted CD34+ cells from the peripheral blood of a wholesome donor (aged 20–24). The CT27 patient-derived trophoblast stem cell line used on this examine had been derived from placental cytotrophoblast cells and was obtained from the RIKEN stem cell financial institution (RCB4936:CT27). Human placentas had been obtained from wholesome ladies with signed knowledgeable consent of the donors, and the approval of the Ethics Committee of Tohoku College College of Drugs (analysis license 2014-1-879).

Statistics and reproducibility

Graphs and statistical analyses had been made in GraphPad Prism and Origin 2022. Pattern sizes are specified within the determine captions, the place n refers back to the variety of distinct samples. For the cell viability assay, a one-way evaluation of variance (ANOVA) check with a Tukey publish hoc check was carried out (n = 3, df = 4). For the blood compatibility assessments, a one-way ANOVA with a Holm–Sidak publish hoc check was carried out (n = 3, df = 6). Knowledge are expressed as imply ± s.d., with *P < = 0.05, **P < = 0.01 and ***P < = 0.001 indicating statistical significance. No statistical technique was used to find out pattern dimension, and no knowledge had been excluded from the analyses. The experiments weren’t randomized, and the investigators weren’t blinded to allocation throughout experiments and consequence evaluation.

Reporting abstract

Additional data on analysis design is offered within the Nature Portfolio Reporting Abstract linked to this text.