Polytron pt 3100 homogenizer

tHIOs were homogenized with a Polytron PT3100 homogenizer (Kinematica). RNA was then extracted from the tHIOs using the E.Z.N.A Total RNA kit I (Omega Bio-Tek) according to the manufacturer’s instructions. Take3 Micro-Volume Plate (BioTek) in a Cytation 5 reader (BioTek) was used to determine RNA concentration and purity. The cDNA was synthesized using an iScript reverse transcription kit (BIO-RAD) with a Mastercycler (Eppendorf). RT-qPCR was performed using PowerUp SYBR Green Master Mix (ThermoFisher Scientific) according to the manufacturer’s instructions for the fast protocol on a CFX Touch Real-time PCR Detection System (BIO-RAD). Primers were obtained from Integrated DNA Technologies and are listed in Table 1.

Heparin microparticles were fabricated as previously described (37 (link),38 (link)). Briefly, EDC/Sulfo-NHS chemistry was used to substitute methacrylamide groups on heparin. Heparin methacrylamide was then dissolved in phosphate-buffered saline (PBS) and mixed with equimolar amounts of the free radical initiators, ammonium persulfate (Sigma Aldrich) and tetramethylethylenediamine (TEMED, Sigma Aldrich). A water-in-oil emulsion was then formed by adding the heparin solution dropwise into 60mL of corn oil and 1mL of polysorbate 20 (Promega) and then homogenized on ice for 5 min at 3000rpm (Polytron PT3100 Homogenizer, Kinematica). Free radical polymerization and thermal cross-linking of the methacrylamide groups was carried out by immersing the emulsion in a 55˚C water bath under constant stirring and nitrogen purging for 30 minutes. The HMPs were collected following centrifugation for 10 minutes at 3000rpm and subsequently washed in acetone, deionized water several times, and 70% ethanol for sterilization. HMPs were lyophilized and stored at 4˚C until ready for incorporation into the alginate constructs. HMPs were characterized following fabrication and confirmed to retain their functionality with evaluation of growth factor binding and release kinetics (38 (link)).

The tHIOs were homogenized with a Polytron PT3100 homogenizer (Kinematica). RNA was then extracted from the tHIOs using the E.Z.N.A Total RNA kit I (Omega Bio-Tek) according to the manufacturer’s instructions. A Take3 Micro-Volume Plate (BioTek) in a Cytation 5 reader (BioTek) was used to determine RNA concentration and purity. A minimum quantity of 200 ng of RNA with a 260/280 ratio of 1.8–2.2 was included in analysis. The cDNA was synthesized using an iScript reverse transcription kit (BIO-RAD) with a Mastercycler (Eppendorf). RT-qPCR was performed using PowerUp SYBR Green Master Mix (ThermoFisher Scientific) according to the manufacturer’s instructions for the fast protocol on a CFX Touch Real-time PCR Detection System (BIO-RAD). Primers were obtained from Integrated DNA Technologies and are listed in Supplemental Table 3.

HMPs were fabricated as previously described [32 (link)] and as schematized in Figure 1A. Briefly, 55.6 mg of heparin methacrylamide, 18 mM ammonium persulfate (APS; Sigma Aldrich), and 18 mM tetramethylethylenediamine (TEMED; Sigma Aldrich) were dissolved in phosphate buffered saline (PBS; Corning Mediatech, Manassas, VA). 500 μL of heparin solution was homogenized for 5 minutes at 3000 rpm into a mixture of 60 mL of corn oil (Mazola) and 1 mL of polysorbate 20 (Promega, Madison, WI) using a Polytron PT3100 homogenizer (Kinematica, Lucerne, Switzerland). The emulsion was immersed in a 55°C water bath, and the free radical cross-linking reaction proceeded under constant stirring and nitrogen purging for 30 minutes. The solution was centrifuged for 10 minutes at 3000 rpm, and the resulting HMP pellet was washed once in acetone and several times in ddH₂O, prior to being disinfected in 70% ethanol for 30 minutes. The HMPs were lyophilized (Labconco, Kansas City, MO) and stored at 4°C until use.