Short-Stranded Zein Fibers for Muscle Tissue Engineering in Alginate-Based Composite Hydrogels

Abstract

Cultivated meat is a nascent technology that aims to create an environmentally and animal-friendly alternative to conventional meat. Producing skeletal muscle tissue in an animal-free system allowing for high levels of myofusion and maturation is important for the nutritional and sensorial value of cultivated meat. Alginate is an attractive biomaterial to support muscle formation as it is food-safe, sustainable and cheap and can be crosslinked using non-toxic methods. Although alginate can be functionalized to promote cell attachment, limitations in its mechanical properties, including form, viscosity, and stress relaxation, hinder the cellular capacity for myogenic differentiation and maturation in alginate-based hydrogels. Here, we show that the addition of electrospun short-stranded zein fibers increased hydrogel degradation, resulting in faster compaction, improved cell-gel interaction, and enhanced alignment of bovine muscle precursor cells. We conclude that fiber-hydrogel composites are a promising approach to support optimal formation of 3D constructs, by improving tissue stability and thus prolonging culture duration. Together, this improves muscle-related protein content by facilitating myogenic differentiation and priming muscle organoids for maturation.

Keywords: alginate hydrogel; cultivated meat; cultured meat; electrospinning; fiber-hydrogel composites; muscle tissue; tissue engineering; zein.

Figure 1

Schematic overview of fabrication and incorporation of zein short-stranded fibers (SSFs) into RGD–alginate BAMs.

Figure 2

Morphology of electrospun zein fibers. Scanning electron microscopy (SEM) images showing morphology of electrospun fibers using 20, 25, 28, 30, and 35 wt% zein solution. Scale bars, 10 µm.

Figure 3

Cell attachment and processing of electrospun fibers. (A) Scanning electron microscope (SEM) images showing cell attachment to zein fibers. Scale bar, 10 µm. (B) Fluorescence microscopy images of satellite cells attaching to zein fibers. Phalloidin, white; Hoechst, blue. Scale bar, 100 µm. (C) SEM images showing the morphology of aligned and non-aligned zein fibers before processing. Scale bar, 50 µm. (D) Brightfield microscopy images of zein fibers shown in C, post-processing with ultrasonication. Scale bars, 75 µm.

Figure 4

Characterization of alginate hydrogels with and without electrospun zein fibers. (A) Hydrogel stiffness on day 0 with 0, 0.1, and 0.5 wt% zein-SSFs without cells. Error bars indicate s.d., n = 4. (B) Swelling of hydrogels on days 0, 1, and 3 with 0, 0.1 and 0.5 wt% zein-SSFs. Error bars indicate s.d., n = 3. (C) Relative remaining mass of hydrogels on days 0, 1 and 3 with 0, 0.1 and 0.5 wt% zein-SSFs. Error bars indicate s.d., n = 3. (D) Scanning electron microscopy (SEM) images showing the structure of alginate hydrogels with 0, 0.1, and 0.5 wt% zein-SSFs. Scale bar, 500 µm. (E) SEM images with increased magnification showing fiber morphology inside alginate hydrogels with 0, 0.1, and 0.5 wt% short-stranded zein fibers. Arrowheads indicate the presence of zein-SSFs. Scale bar, 10 µm. p-values; * p ≤ 0.05, p ≤ 0.01, *** p ≤ 0.001.

Figure 5

The blending of short-stranded zein fibers improves compaction during cultured muscle formation. (A) BAM formation around stainless steel pillars after 1 and 7 days culture with 0, 0.1 and 0.5 wt% short-stranded zein fibers. Arrowheads indicate the position of the BAMs around the pillar. Scale bar, 3 mm. (B) Quantification of images shown in (a) with additional timepoints. Degree of compaction on days 1, 3, 5 and 7. Error bars indicate s.d., n = 4. (C) Fluorescent images showing BAM morphology on days 3, 7 and 10. Phalloidin, red; zein, yellow; Hoechst, blue. Scale bar, 100 µm. p-values; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 6

Protein yield and metabolic changes after zein fiber addition. (A) Total protein measurements for BAMs with 0, 0.1 and 0.5 wt% zein fibers at indicated timepoints. Error bars indicate s.d., n = 2. No statistically significant differences were observed, with P-values > 0.05. (B) Desmin ELISAs in BAMs with 0, 0.1 and 0.5 wt% zein fibers. Error bars indicate s.d., n = 2. p-values > 0.05. (C) Same as B, but slow myosin heavy-chain ELISAs. Error bars indicate s.d., n = 2. P-values > 0.05. (D) Western blot of selected muscle-related proteins during myogenic differentiation, analyzed at indicated timepoints. (E) Lactate concentrations recorded in the culture medium at indicated timepoints. Values are normalized to day 0. p-values > 0.05. (F) As E, but for glucose concentrations. (G) As E, but for ammonia concentrations. (H) As E, but for glutamax concentrations.