Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production

Abstract

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment-with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Fig. 1. C2C12 metabolic activity expressed as percent reduction of alamarBlue (AB) on different fungal species at 48 h after seeding.

No microcarrier as the negative control and nonedible carrier Cytodex as the positive control. Superscript letters (a–c) indicate statistically significant homogenous groups differing in the parameters among the strains (p < 0.05, F-test). Error bars represent standard deviations.

Fig. 2. C2C12 on carriers at differing seeding densities.

C2C12 transfected with TdTomato (red fluorescent) on Cytodex, Mycelium carrier (inactivated A. oryzae 1), and no microcarrier (MC) at 72 hours after seeding. Scale bar is 281 μm.

Fig. 3. C2C12 metabolic activity represented as % reduction of alamarBlue (AB) on Cytodex, mycelium carriers and no microcarrier (MC) at 24 h from time of seeding (represented in lighter colored bars) and 72 h from time of seeding (represented in darker color bars).

* indicates significant differences at p ≤ 0.05 with n ≥ 3 and ns indicates no significant differences. Error bars represent standard deviations.

Fig. 4. Relative expression of differentiation markers PAX7, MYOD1, MYOG, MYH2 for C2C12 at four different sampling time points on cytodex and mycelium carriers.

Samples were collected right after switching to differentiation media (time point 0, T0) and at 48 hours (time point 1, T1), 96 h (time point 2, T2), and 168 h (time point 3, T3). * indicates significant differences at p ≤ 0.05, ** at p ≤ 0.01, **** at p ≤ 0.0001, with n indicated by individual data points and ns indicating no significant differences. Plate wells that did not amplify are not shown. Error bars represent standard deviation.

Fig. 5. Bovine satellite cells on carriers.

a bSC immunostained with Hoechst (blue fluorescent) on Cytodex microcarriers and mycelium carriers after 72-h incubation seeded at 5 × 10⁵ cells/mL. Dotted lines represent the edge of the pellet (mycelium carriers). b bSC metabolic activity represented as % reduction of alamarBlue (AB) on Cytodex, mycelium carriers and no microcarrier (MC) at 24 h from time of seeding (represented in lighter colored bars) and 72 h from time of seeding (represented in darker color bars). * indicates significant differences at p ≤ 0.05 with n ≥ 3 and ns indicates no significant differences. Error bars represent standard deviations. Scale bar is 130 μm.

Fig. 6. Relative expression of differentiation markers MYOD1, MYOG, MYH1/2, and MYH3 for bSC at three time points on Cytodex and Mycelium carriers.

Samples were collected 48 h (time point 1, T1), 96 h (time point 2, T2), and 168 h (time point 3, T3). * indicates significant differences at p ≤ 0.05, ** at p ≤ 0.01, **** at p ≤ 0.0001, with n indicated by individual data points and ns indicates no significant differences. Plate wells that did not amplify are not shown. Error bars represent standard deviation.