lm 111, by Bio-Techne - Product details

1

Fabrication of Biomimetic Gelatin-Collagen Sponges

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Example 1

In this Example, biomimetic sponges were fabricated.

A 3 wt. % porcine skin gelatin (Sigma-Aldrich) solution was prepared in DI water heated to 60° C. After the gelatin had completely dissolved, the solution was allowed to cool to 50° C., and EDC (20 mM) was added. The solution was then vortexed vigorously and added to square plastic molds with 3 mg/mL rat tail collagen I (Gibco) solution in gelatin:collagen ratios of 100:0 (pure gelatin), 90:10, and 70:30. LM-111 (Trevigen) was then added to the molds at a final concentration of 50 μg/mL and mixed thoroughly by pipetting the solution in the molds up and down. The molds were placed in a 100% methanol bath, and allowed to gel at 4° C. for 30 minutes, followed by overnight freezing at −8° C. The molds were then moved to a −80° C. freezer for 24 hours. Then the methanol was removed and they were frozen for another 24 hours at −80° C. The frozen molds were lyophilized for at least 19 hours. The cross-sections of the lyophilized sponges were observed through scanning electron microscopy (SEM) (see, FIGS. 1A-1F). The addition of collagen increased the overall porous structure and void space in the sponges.

US11744922B2. Biomimetic sponges for tissue regeneration (2023-09-05). Saint Louis University [US]. Inventors: Koyal Garg [US], Gabriel Haas [US].

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2

Fabrication of Gelatin-Collagen Sponges

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A 3 wt% porcine skin gelatin (Sigma–Aldrich) solution was prepared in deionized (DI) water and heated to 60°C. After the gelatin had completely dissolved, the solution was allowed to cool to 50°C. The cooled gelatin solution was combined with 20 mM of EDC. Rat tail collagen I (Gibco, 3 mg/ml) was mixed with the gelatin solution at a ratio of 70:30 by volume. LM-111 (Trevigen) were added to the solution at final concentrations of 50 μg/ml. FK-506 (Abcam) was added at a final concentration of 25 μM, respectively. The solution was then aliquoted into a 48-well plate at 700 μl/well. The well plate was placed in a 100% methanol bath, allowing the sponges to gel at 4°C for 30 min, followed by overnight freezing at −8°C. The well plate was then removed from the bath and moved to a − 80°C freezer for 48 hr and subsequently lyophilized for at least 12 hr. Prior to in vitro culture or surgical implantation, the sponges were disinfected using ethanol and rinsed twice in sterile 1× phosphate-buffered saline (PBS).

Haas G., Dunn A., Madsen J., Genovese P., Chauvin H., Au J., Ziemkiewicz N., Johnson D., Paoli A., Lin A., Pullen N, & Garg K. (2021). Biomimetic sponges improve muscle structure and function following volumetric muscle loss. Journal of biomedical materials research. Part A, 109(11), 2280-2293.

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3

Laminin-111 Functionalization for Cell Culture

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PEGLM was prepared as previously described (Fearing et al., 2019 (link); Francisco et al., 2013 (link)). Laminin-111 (LM-111, Trevigen; Gaithersburg, MD, USA) was reacted with acrylate-PEG-hydroxysuccinimide (Ac-PEG-NHS, 10 kDa, Creative PEGWorks; Winston-Salem, NC, USA) in order to form PEGLM. This solution was then dialyzed against PBS in order to remove unreacted Ac-PEG-NHS groups and PEGLM concentration was determined at 280 nm absorbance. Stiff (> 1 GPa) tissue culture surfaces (i.e., polystyrene well plates or glass chamber slides; Nunc Lab-Tek Chamber Slide Systems, Thermo Fisher Scientific; Waltham, MA, USA) were coated with the PEGLM solution (diluted with sterile PBS to a working concentration of 22.5 μg/mL). The PEGLM was allowed to adsorb to the culture surface overnight at 4 °C. The following day, the solutions were removed, and surfaces were rinsed with sterile PBS prior to inception of the experiment.

Speer J., Barcellona M., Jing L., Liu B., Lu M., Kelly M., Buchowski J., Zebala L., Luhmann S., Gupta M, & Setton L. (2021). INTEGRIN-MEDIATED INTERACTIONS WITH A LAMININ-PRESENTING SUBSTRATE MODULATE BIOSYNTHESIS AND PHENOTYPIC EXPRESSION FOR CELLS OF THE HUMAN NUCLEUS PULPOSUS. European cells & materials, 41, 793-810.

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4

PDMS Mold Fabrication for Collagen and LM Coating

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Polydimethylsiloxane (PDMS) molds (4 cm^{2 (link)}) were autoclaved before use. Rat tail collagen (COL) I (Gibco, 9.33 mg/mL) and LM-111 (Trevigen, 6 mg/mL) solution was diluted to 200 μg/mL using 1× phosphate-buffered saline (PBS). A total of 500 μL of the diluted COL and LM-111 solution was added to PDMS molds. The molds were incubated at 37°C for 1 h, after which the collagen and LM solutions were removed and rinsed in 1 × PBS before cell seeding. PDMS molds were used with the C-Pace E-stim system as per manufacturer's instructions. PDMS is typically chosen for such studies due to its (1) biocompatibility, (2) transparency for visualization of cells under a microscope, and (3) high adsorption capacity that permits coating with various ECM proteins such as collagen and LM.^{35 (link)}

Patel A., Vendrell-Gonzalez S., Haas G., Marcinczyk M., Ziemkiewicz N., Talovic M., Fisher J.S, & Garg K. (2019). Regulation of Myogenic Activity by Substrate and Electrical Stimulation In Vitro. BioResearch Open Access, 8(1), 129-138.

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5

Laminin-111 Functionalization for Cell Culture

Cited 2 times

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PEGLM was prepared as previously described (Fearing et al., 2019 (link); Francisco et al., 2013 (link)). Laminin-111 (LM-111, Trevigen; Gaithersburg, MD, USA) was reacted with acrylate-PEG-hydroxysuccinimide (Ac-PEG-NHS, 10 kDa, Creative PEGWorks; Winston-Salem, NC, USA) in order to form PEGLM. This solution was then dialyzed against PBS in order to remove unreacted Ac-PEG-NHS groups and PEGLM concentration was determined at 280 nm absorbance. Stiff (> 1 GPa) tissue culture surfaces (i.e., polystyrene well plates or glass chamber slides; Nunc Lab-Tek Chamber Slide Systems, Thermo Fisher Scientific; Waltham, MA, USA) were coated with the PEGLM solution (diluted with sterile PBS to a working concentration of 22.5 μg/mL). The PEGLM was allowed to adsorb to the culture surface overnight at 4 °C. The following day, the solutions were removed, and surfaces were rinsed with sterile PBS prior to inception of the experiment.

Speer J., Barcellona M., Jing L., Liu B., Lu M., Kelly M., Buchowski J., Zebala L., Luhmann S., Gupta M, & Setton L. (2021). INTEGRIN-MEDIATED INTERACTIONS WITH A LAMININ-PRESENTING SUBSTRATE MODULATE BIOSYNTHESIS AND PHENOTYPIC EXPRESSION FOR CELLS OF THE HUMAN NUCLEUS PULPOSUS. European cells & materials, 41, 793-810.

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Lm 111

Lab products found in correlation

5 protocols using lm 111

Fabrication of Biomimetic Gelatin-Collagen Sponges

Fabrication of Gelatin-Collagen Sponges

Laminin-111 Functionalization for Cell Culture

PDMS Mold Fabrication for Collagen and LM Coating

Laminin-111 Functionalization for Cell Culture

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