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In Vitro Insect Muscle for Tissue Engineering Applications


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Tissue engineering is primarily associated with medical disciplines; thus, research has focused on mammalian cells. For applications where clinical relevance is not a constraint, it is imperative to evaluate the potential of alternative cell sources to form tissues in vitro. Specifically, skeletal muscle tissue for bioactuation and cultured foods could benefit from the inclusion of invertebrate cells, due to less stringent growth requirements and versatile features related to biomass. Here, we used a Drosophila muscle cell line to demonstrate the benefits of insect cells relative to those derived from vertebrates. The cells were adapted to serum-free media, transitioned between adherent and suspension cultures, and manipulated via hormones. Furthermore, we analyzed scaffolds to support muscle growth and assayed cellular protein and minerals to evaluate nutrition potential. The insect muscle cells exhibited advantageous growth patterns and hold unique functionality for tissue engineering applications beyond the medical realm.
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In Vitro Insect Muscle for Tissue Engineering Applications
Natalie R. Rubio,
Kyle D. Fish,
Barry A. Trimmer,
and David L. Kaplan*
Department of Biomedical Engineering, Tufts University, Science & Technology Center, 4 Colby Street, Medford, Massachusetts
02155, United States
Department of Biology, Tufts University, 200 Boston Avenue #4700, Medford, Massachusetts 02155, United States
SSupporting Information
ABSTRACT: Tissue engineering is primarily associated with
medical disciplines, and research has thus focused on
mammalian cells. For applications where clinical relevance is
not a constraint, it is useful to evaluate the potential of
alternative cell sources to form tissues in vitro. Specically,
skeletal muscle tissue engineering for bioactuation and cultured
foods could benet from the incorporation of invertebrate cells
because of their less stringent growth requirements and other
versatile features. Here, we used a Drosophila muscle cell line to
demonstrate the benets of insect cells relative to those derived
from vertebrates. The cells were adapted to serum-free media,
transitioned between adherent and suspension cultures, and
manipulated with hormones. Furthermore, we analyzed
edible scaolds to support cell adhesion and assayed cellular protein and minerals to evaluate nutrition potential. The insect
muscle cells exhibited advantageous growth patterns and hold unique functionality for tissue engineering applications beyond
the medical realm.
KEYWORDS: insect cell culture, skeletal muscle tissue engineering, chitosan scaolds, bioactuation, cellular agriculture, cultured meat
Advances in tissue engineering have driven the emergence of
new products and industries beyond the realm of regenerative
medicine, including organs-on-a-chip, soft robotics, and
biofabricated food and materials. Specically, skeletal muscle
tissue engineering is now being applied for the development of
muscle-powered biobots and bioengineered meat, also known
as cultured meat.
These applications are not constrained by
concerns of immunogenicity, host-integration, or in vivo-like
function. Instead, relevant challenges include lowering costs
and achieving ecient, large-scale production so that tissue
engineered commodities can be competitive against their
conventional counterparts (e.g., electrical actuators and
agriculturally farmed meat).
Muscle tissue produced as food
should also be visually and texturally similar to farmed meat,
appealing to consumers and nutritionally advantageous.
Conversely, bioactuators should generate large contractile
force, operate under a range of environmental conditions, and
incorporate control systems.
Insect cells are potentially better
suited than mammalian cells to address many of these
Commonly used cells for skeletal muscle tissue research
include the mouse myoblast cell line C2C12, the rat myoblast
cell line L6, and human cells obtained from primary lines or
induced pluripotent stem cells.
These cell types are typically
grown as adherent cultures at 37 °C with 5% carbon dioxide in
sodium bicarbonate-buered basal medium supplemented with
fetal bovine serum. These conditions, while feasible for bench-
scale culture, create hurdles for achieving cost-ecient
production at scale for commercial cell-based products.
Specically, animal serum is costly and inconsistent, above
ambient incubation temperatures require increased energy use,
and adherent cell lines need complex substrates (e.g.,
microcarriers, hollow bers) for high density growth in
bioreactor systems.
In contrast, many insect cell lines are able to transition
between adherent and suspension culture, and are best suited
for temperatures within the ambient range of 1930 °C and
slightly acidic pH levels (6.26.4).
Unlike vertebrate cells,
insect cells can be grown in a nonhumidied environment and
do not require carbon dioxide exchange.
It is also reported to
be relatively simple to adapt insect cells to both serum-free
medium and suspension culture.
Furthermore, immortal or
continuous insect cell lines are straightforward to obtain
compared to vertebrate species, and many lines have been
observed to retain their phenotype after over 120 cell
In our prior study of Manduca sexta, primary
cells maintained viability in culture for a month without media
This set of unique culture characteristics makes
insect cells a particularly promising platform for novel
Received: October 14, 2018
Accepted: January 2, 2019
Published: January 2, 2019
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applications of tissue engineering, and could contribute to
scalable, cost-eective production systems for bioactuators and
Bioactuators are theorized to be advantageous over conven-
tional actuators because of their eciency and sustainability,
and their capacity for self-assembly, self-healing, and
Most bioactuator research at cellular and
tissue levels has focused on mammalian cardiac or skeletal cells
or insect dorsal vessel tissue (DVT) explants.
The general
research strategy is to couple cultured cells or tissues with
scaold systems to perform mechanical work. For example,
cardiomyocyte cell sheets have been combined with poly-
dimethylsiloxane (PDMS) constructs to create a micropump
with linear ow rates of 2 nL/mL.
PDMS molds have
similarly been used with skeletal muscle cells to create
microtissues under optogenetic control.
Insect tissue explants
have also been of interest due to their tolerance of temperature
uctuations. Explanted DVT has been attached to PDMS
molded devices which were operable at room temperature and
generated 20 μN force.
In our own studies, we used cells
isolated from Manduca sexta embryos to make simple muscle
ber bioactuators that contracted under a wide range of
temperature, pH, and nutrient conditions.
Cultured meat is another innovation derived from advances
in tissue engineering. By producing meat from cell cultures
rather than whole organisms (e.g., farm animals), it is emerging
as a potential solution to global food issues. Cultured muscle
tissue production generally consists of (1) obtaining cells from
an immortalized line or biopsy isolation, (2) proliferating the
cells at scale in a serum-free media, and (3) dierentiating the
cells into muscle on an edible, degradable or reusable scaold.
Research in this eld has gained traction since 2013, when a
cultured beef burger proof of conceptwas produced.
Skeletal muscle development from porcine induced pluripotent
stem cells has since been reported, oering a potentially
reliable farm animal-derived cell line for food technology.
Primary muscle cell lines from chicken, cow, pig, and horse
have also been isolated and dierentiated in vitro.
progress has been made on the development of sustainable and
edible scaold systems. For example, cellulose-based scaolds
fabricated from decellularized plants (e.g., spinach, apples)
have been shown to support mammalian muscle growth.
Reliable cell sources, serum-free media formulations, and 3D
scaold systems must be developed in order for insect muscle
tissue engineering to be applied for bioactuator and cultured
meat development. To date, in vitro insect muscle research has
utilized explants or primary cells isolated from insect tissue.
However, primary cell isolates result in mixed cell cultures and
require frequent and labor-intensive isolation procedures.
Drosophila melanogaster adult muscle immortalized progenitor-
like cell line may be a promising initial cell source for insect
tissue engineering because the cells (1) express GFP for ease of
imaging, (2) are highly proliferative, and (3) can dierentiate
upon treatment with the insect molting hormone 20-
It is important for the culture medium
to be serum-free, low-cost, and support muscle growth and
dierentiation. Fortunately, there are many commercial and
homemadeserum-free media formulations available for
insect cells which can be veried or adapted to support
muscle-specic cells.
There is also a need for the design of
aordable scaold systems capable of supporting 3D insect
muscle constructs. Mushroom-derived chitosan is a promising
biomaterial for development of such scaolds, as it is easily
accessible, edible, widely used in tissue engineering, and
already incorporated in food products as an additive or dietary
The combination of stable insect muscle cell
lines, optimized media, and scaolding techniques will allow
for further evaluation and analysis of the potential applications
of insect muscle tissue engineering.
The objective of the present work was to evaluate the
potential of D. melanogaster muscle cells to serve as a platform
for tissue engineering applications. To accomplish this goal, we
assessed features relevant to scaleable production (e.g., serum-
free culture, suspension culture, hormone regulation schemes),
analyzed 3D culture systems using mushroom-derived chitosan
scaolding, and quantied cellular levels of protein and
minerals for nutritional insight.
Materials and Methods. Cell Culture. Drosophila melanogaster
adult muscle progenitor-like cells (DrAMPCs) were acquired from
Kerafast (Boston, MA) (#EF4006). The cell line was originally
immortalized by the Persimmon Research Group at Harvard
University, and it was derived from primary embryo cultures in
which Gal4 drives RasV12 and GFP expression. DrAMPCs were
cultured in insect growth media composed of Schneiders Insect
Medium from Sigma-Aldrich (St. Louis, MO) (#S0146) supple-
mented with 10% heat inactivated fetal bovine serum from
ThermoFisher (Waltham, MA) (#16140) and 1% penicillin/
streptomycin (ThermoFisher, #15140122). For serum-free growth
media experiments, media consisted of Ex-Cell 405 Serum-Free
Medium (Sigma-Aldrich, #14405C) and 1% penicillin/streptomycin.
For static culture, DrAMPCs were cultured in either plasma-treated
asks (ThermoFisher, #156499) or ultra-low attachment asks
(Corning, NY) (#CLS3814) and incubated at 19 °Cina
temperature-controlled incubator from VWR (Radnor, PA)
(#89511416). For suspension culture, DrAMPCs were cultured in
shaker asks (ThermoFisher, #41150250) on an orbital shaker set at
40 rpm at room temperature. When indicated, dextran sulfate (Sigma-
Aldrich, #67578) was supplemented to serum-free media by
dissolution and sterile ltration with 0.2 μm bottle top lters
(ThermoFisher, #5953320). For hormone treatment and dier-
entiation experiments, DrAMPCs were cultured in insect growth
media or serum-free media supplemented with methoprene (Sigma-
Aldrich, #33375) and/or 20-hydroxyecdysone (Sigma-Aldrich,
#H5142) which were dissolved in DMSO (Sigma-Aldrich,
#D8418). DrAMPCs were seeded at 75 000 or 300 000 cells/cm2in
2D culture and 1 000 000 cells/sponge in 3D culture. For mammalian
controls, C2C12 cells from ATTC (Manassas, VA) (#CRL-1772)
were cultured in growth media composed of DMEM + Glutamax
(ThermoFisher, #10566) supplemented with 10% heat inactivated
fetal bovine serum and 1% penicillin/streptomycin. C2C12s were
seeded at 10,000 cells/cm2.
Adhesion, Proliferation, and Viability Assays. Cell adhesion to
tissue culture plastic and chitosan lms was quantied via a MTS
Assay from Promega (Madison, WI) (#G3582) by measuring
absorbance after 2.5-h incubation with the MTS reagent. Cell
proliferation and viability were measured by uorescence, Live/Dead
kit (ThermoFisher, #L3224) stained image analysis or CyQuant
proliferation assays (ThermoFisher, #C7026). For uorescence
measurements, GFP-expressing DrAMPCs were imaged on a
uorescence microscope over the course of a week using the
automated multipoint capture feature. Fluorescence was quantied on
Fiji software and normalized to the values determined at the rst time-
point. For Live/Dead stained image analysis, cells were stained
following the kit protocols and imaged on a uorescence microscope.
The images were analyzed with Fiji to quantify the viability and total
cell population over time. For CyQuant proliferation assays, cells were
plated in 96-well plates for each time-point. At each time-point, media
was blotted from the plates and plates were stored at 80 °C. After all
time points were collected, plates were thawed to room temperature
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and stained with CyQuant working solution for 5 min. Microplate
measurements were performed on a SpectraMax M2 reader from
Molecular Devices (San Jose, CA). Cell populations were determined
from a standard curve.
Staining and Imaging. Cell viability was determined by a Live/
Dead staining kit (ThermoFisher, #L3224). At time of assay, media
was gently aspirated from the cell surface and cells were rinsed with
phosphate buered saline (PBS) (ThermoFisher, #14040133). Cells
were stained with 2 μM calcein AM and 4 μM EthD-1 solution for 30
min at room temperature in the dark and imaged with a uorescence
microscope. For immunocytochemical staining, media was gently
aspirated from cells, and the cell surface was rinsed with PBS. Cells
were xed with 4% paraformaldehyde from Fisher Scientic
(Hampton, NH) (#J61899AK) for 10 min and rinsed with PBS.
Cells were then permeabilized with 0.5% Triton X-100 and rinsed
with blocking buer consisting of phosphate buered saline, 5% fetal
bovine serum, and 0.05% sodium azide. Cells were incubated in
blocking buer for 15 min before addition of primary antibody, then
incubated at 4 °C overnight. Cells were rinsed and incubated with
fresh blocking buer for 15 min before addition of secondary
antibody, then incubated in the dark on ice for 1 h. Again, cells were
rinsed and incubated with fresh blocking buer for 15 min before
counterstaining and mounting with DAPI mounting medium from
Abcam (Cambridge, U.K.) (#ab104139). Detailed antibody informa-
tion is available in Table SI2. Fluorescence imaging was performed on
a Keyence microscope (Osaka, Japan) (#BZ-X700). Confocal imaging
was performed on a TCS SP8 microscope from Leica Microsystems
(Wetzlar, Germany).
Scaold Fabrication. Mushroom chitosan of 100 kDa molecular
weight from Chinova Bioworks (New Brunswick, Canada) was
dissolved in 2% acetic acid in distilled water for 12 h at room
temperature on a stir plate. The chitosan solution was centrifuged at
16 880gfor 3 h to remove undissolved particles and diluted in distilled
water to the desired concentrations (1%, 2% and 4%). Concentrations
were veried by drying small volumes at 60 °C for 2 h and calculating
dry weight/wet weight. To prepare lms, the solution was cast on
plastic and allowed to dry overnight. To prepare sponges, the solution
was poured into PDMS molds with an aluminum sheet separating one
side of the mold from a separate chamber. Liquid nitrogen was poured
into the chamber opposite the chitosan solution and continually
replenished until the entire solution had frozen across the temperature
gradient. Samples were then lyophilized for 48 h. Prior to sterilization
and seeding, lms, and sponges were submerged in 1 M sodium
carbonate for 1 h at room temperature and subsequently rinsed three
times in distilled water for 20 min and soaked in distilled water
overnight. For hydrated mechanical analysis, sectioned sponges were
soaked in PBS prior to testing.
Cell Seeding. Sponges used for seeding were cylinders 6 mm in
diameter and 1.5 mm thick. They were sterilized for 24 h with 70%
ethanol and UV exposure, and then soaked in growth media
overnight. They were then seeded with 1 000 000 DrAMPCs in 50
μL of media and incubated for 4 h before 1 mL of media was added to
each well. Films were cast in 24-well plates and seeded at medium
(75 000 DrAMPCs/cm2) or high (300 000 DrAMPCs/cm2). Media
changes were performed once per week.
Mechanical Testing. Compression tests were performed on an
Instron 3366 from TA Instruments (New Castle, TE) with a strain
rate of 1 mm/min to a total strain of 30%, and modulus values were
calculated for the 2%10% compression interval. Samples were tested
in the hydrated state (immersed in 1×PBS). Sponge dimensions used
were 8 ×8×8 mm cubes.
Nutrition Testing. DrAMPC and C2C12 cells were cultured and
harvested into aliquots of 20 million and 10 million cells respectively
for nutritional analysis. Cells were lysed with RIPA buer (Thermo-
Fisher, #89900) and 1% Halt Protease Inhibitor Cocktail EDTA-Free
(ThermoFisher, #78425). Protein was quantied by a Pierce BCA
Protein Assay Kit (ThermoFisher, #23225) according to manufac-
turer instructions. Iron and zinc were quantied by an Iron Assay Kit
(Abcam, #ab83366) and Zinc Assay Kit (Abcam, #ab102507)
according to manufacturer instructions. For iron fortication
experiments, cells were cultured with 10% Iron-Fortied Bovine
Serum (Sigma-Aldrich, #12138C). Microplate measurements were
performed on a SpectraMax M2 reader (Molecular Devices).
Thermogravimetric Analysis. Thermogravimetric analysis with
ramped heat was performed on samples of chitosan sponge with a
Thermogravimetric Analyzer (TA Instruments, #Q500). Samples
were heated from room temperature to 500 °C at a rate of 20 °C per
Statistical Analysis. Statistical analysis was performed with
GraphPad Prism 7.04 software. Error bars in column charts are
standard deviations. Statistical signicance was determined via two-
way ANOVA and multiple comparisons with the Sidak posthoc test or
via multiple ttests with the Holm-Sidak posthoc test with alpha =
0.05. Additional info as well as the p-values for each statistically
signicant comparison are listed in Table SI3.
Adaptation of Insect Muscle Cells to Serum-Free
Media and Inducing Single-Cell Suspension Culture. A
primary goal of our research was to adapt insect muscle cells to
serum-free media. We adapted the cells either immediately
from 0% to 100% ExCell 405 Serum-Free Insect Medium
(EC405) (Figure 1b), or gradually over the course of 2 weeks
by passaging the cells in increasing concentrations of EC405
(Figure 1c). A subset of cells was maintained in serum-
supplemented media as a control (Figure 1a). Details of the
adaptation schedule are listed in Table SI1. The immediately
adapted cells initially proliferated at rates equivalent to the
control cells; however, after the rst 48 h, the growth rate
decreased. After 1 week in culture, the growth of immediately
adapted cells was stagnant, and the cell morphology appeared
more neuron-like than myoblast-like; with multiple cell
extensions protruding from the cell body (Figure 1b). The
gradually adapted cells retained their myoblast-like morphol-
ogy identied by slight elongation (Figure 1c) and exhibited
comparable growth rates to controls (Figure 1d). As shown in
Figure 1d, the insect muscle cells appeared to exhibit diauxic
growth. The two growth phases are distinguished by adherent
growth and suspension growth. At low to medium cell
densities, the cells are adherent. They proliferate until the
surface is overconuent (growth phase 1) and subsequently
begin growing in suspension (growth phase 2).
Once the DrAMPC culture exhibited steady growth in
EC405, the monolayer culture was transitioned to suspension
culture. It was noted that when static culture asks became
overconuent, the cells continued to grow in three-dimen-
sional aggregates or in suspension. When the plasma-treated
culture surface (Figure 1e) was switched with an ultra-low
attachment surface (Figure 1f), the DrAMPCs did not attach
and instead proliferated in suspension, at rst as single cells
and then forming aggregates, reaching a maximum cell density
of 1E6 cells/mL after 5 days. The cells were then transitioned
from static suspension culture to agitated suspension culture
with the use of shaker asks incubated at room temperature. In
agitated suspension, the cells formed aggregates (Figure 1g).
However, the addition of 100 μg/mL dextran sulfate was
sucient to reduce aggregation and promote a single cell
suspension (Figure 1h). After expansion in suspension culture
and removal of dextran sulfate, the cells transitioned back to
adherent monolayers and retained a myoblast-like morphology.
Comparison of Mammalian vs Insect Muscle Cell
Survival and Growth in Starvation Conditions. DrAMPC
cells were noted to be capable of long-term survival and growth
without media refreshment. To investigate this, DrAMPCs and
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C2C12 mouse myoblast cells were cultured in parallel to
compare the survival of mammalian and insect muscle cells in
limited nutrient conditions. Cells were initially fed 5 mL of
media per well in a 6-well plate and subsequently left
undisturbed until time point analysis. The C2C12 cells
decreased in cell viability and total cell number over the
course of 25 days in culture (Figure 2a,b). By day 25, the
majority of cells had detached from the culture surface and
were <20% viable. The DrAMPCs continued to proliferate up
to day 20, after which most of the cells also detached from the
cell surface. However, unlike the C2C12s, the detached
DrAMPCs remained viable, with 70% viability observed after
25 days (Figure 2b,c). Only a small fraction of nonviable cells
Figure 1. Insect muscle cell adaptation to serum-free media and
transition from monolayer to single-cell suspension culture. (a)
Fluorescence microscopy image of DrAMPCs cultured with control
media for 5 days. (b) Fluorescence microscopy image of DrAMPCs
cultured for 5 days after being transferred from control media to
EC405 media. (c) Fluorescence microscopy image of DrAMPCs
cultured for 5 days after being adapted from control media to EC405
media. (d) Growth curve of DrAMPCs cultured in control media or
EC405 after gradual adaptation. The cell population was quantied
from Fiji image analysis and is displayed relative to the cell population
as measured on Day 1. Error bars are standard deviations (n= 5), and
replicates are separate 24-wells. (e) Phase contrast image of
DrAMPCs cultured on a plasma-treated culture surface. (f) Phase
contrast image of DrAMPCs cultured on an ultra-low attachment
culture surface. (g) Fluorescence microscopy image of DrAMPCs
after 1 week in agitated suspension culture in EC405 media. (h)
Fluorescence microscopy image of DrAMPCs after 1 week in agitated
suspension culture in EC405 media supplemented with 100 μg/mL
dextran sulfate.
Figure 2. Comparison of insect and mammalian muscle cell survival
and growth during long-term absence of media refreshment. (a)
Change in C2C12 and DrAMPC populations relative to the
populations determined at Day 5. Populations were quantied from
Fiji image analysis. Error bars are standard deviations (n= 6);
replicates are separate images. (b) Cell viability of C2C12 and
DrAMPC populations as quantied from Fiji analysis of Live/Dead
stained images. Error bars are standard deviations (n= 6); replicates
are separate images. Statistical signicance was determined via the
Holm-Sidak method, with alpha = 0.05. (c) Fluorescence microscopy
images of Live/Dead stained C2C12 and DrAMPC populations over
time. White arrows on Day 25 indicate the cellular aggregates
detached from the culture surface.
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was observed in the center of large cell aggregates. In separate
experiments, DrAMPCs were observed to survive for over two
months without media refreshment. Viability only declined
past 50% when components of the media began to crystallize
as a result of water evaporation (Figure SI4).
Eect of Insect Hormones on Insect Muscle Cell
Proliferation and Dierentiation. DrAMPCs were treated
with methoprene (JH), a juvenile hormone mimic and
insecticide, to investigate the eect of the hormone on cell
proliferation. Cells treated with JH exhibited higher prolifer-
ation than control groups at both day 1 and 5 time points
although the 500 ng/mL treatment level had a larger eect
than the 1000 ng/mL treatment (Figure 3a). Aside from
increasing cell numbers, JH treatment inhibited cell elongation,
a preliminary sign of dierentiation. The average length of JH-
treated (500 ng/mL) cells was 8 μm less than the control cells
(Figure 3b,d).
Cells were also treated with insect molting hormone, 20-
hydroxyecdysone (20-HE). Growth rates of 20-HE treated
cells did not signicantly dier from control groups (Figure 3,
c). Concentrations as low as 40 ng/mL 20-HE were able to
trigger cell elongation, a preliminary sign of dierentiation
(Figure 3, e). When cells were treated with both JH and 20-
HE, cell proliferation increased slightly but not signicantly. It
was noted that the cell population was consistently
heterogeneous, with a fraction of cells elongating and fusing
while the majority of cells remain spherical. To probe for
dierences in the cell population, we stained the cells for
ecdysone receptor (EcdR), the receptor responsible for
binding 20-HE to trigger molting in vivo or dierentiation in
vitro. All cells expressed EcdR, regardless of whether or not
they initiated dierentiation (Figure 4, a).
Induced Insect Muscle Cell Contraction by External
Potassium. The study that generated the DrAMPC line
previously demonstrated that the cells cultured in serum-
supplemented media undergo dierentiation upon treatment
with 20-HE. A small fraction of our EC405 adapted cells were
also triggered by 20-HE to dierentiate and form long,
multinucleated myotubes that express myosin heavy chain; a
skeletal muscle biomarker (Figure 4, b). Spontaneous cell
contractions were not observed during our experiments,
although they have been noted in previous research.
demonstrate the contractile capacity of DrAMPCs, the cultures
were treated with high concentrations of extracellular
potassium (Figure 4b). Intracellular potassium concentrations
in Diptera muscle range between 142 and 179 mM.
The cells
did not contract when treated with 300 mM K+but did
contract when treated with 600 mM K+. The cells were 95%
viable after potassium treatment. Cell fractional shortening was
determined by images analysis. On average, the myoblasts
(elongated cells) contracted by 31.6% ±14.8% (n= 12). In
contrast, the spherical, proliferative precursor cells shortened
by 1.6% ±16.2% (n= 14).
Insect Muscle Cell Growth on Chitosan-Based
Scaolds. Flat lms were fabricated from mushroom-based
chitosan and seeded with DrAMPCs (Figure 5a). After 24 h
and at medium seeding densities, DrAMPCs favored adhesion
to low concentration lms (1%, 2%); however, after multiple
days in culture, all lms became conuent. At high seeding
densities, cells attach to all lms (Figure 5b) after 24 h.
Cellular adhesion was assessed on tissue culture plastic
controls and chitosan lms. On day 1 in culture, the total
cell population was compared to the adherent cell population
by assaying wells as they were (Total) or after being aspirated
and rinsed with PBS (Adherent) (Figure 5c). The total cell
population on tissue culture plastic was greater than the total
Figure 3. Eect of insect juvenile and molting hormones on in vitro
proliferation and dierentiation of insect muscle cells. (a) Cell
population after treatment with 0, 500, or 1000 ng/mL of methoprene
as measured via a CyQuant proliferation assay. Error bars are standard
deviations (n= 5); replicates are separate 96-wells. Statistical
signicance was determined via the Holm-Sidak method, with alpha
= 0.05. (b) Histogram of cell lengths of cultures treated with 0 or 500
ng/mL JH as measured via Fiji image analysis. (c) Cell population
after treatment with 0, 500, or 1000 ng/mL of 20-HE as measured via
a CyQuant proliferation assay. Error bars are standard deviations (n=
5). Dierences between averages were not statistically signicant. (d)
Fluorescence microscopy images of DrAMPCs treated with 0 or 500
ng/mL JH after 4 days in culture. Red circles indicate elongated cells.
(e) Fluorescence microscopy images of DrAMPCs treated with 40
ng/mL 20-HE.
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cell population on the chitosan lms; however, the dierences
in adherent cell populations between conditions were not
statistically signicant. This indicates that cells may grow more
quickly on tissue culture plastic, and the dierence between
total and adherent cells on the plastic may be due to cell
growth in suspension after reaching conuence on the surface.
To investigate the eect of a surface coating on cell adhesion, a
subset of lms was coated with 0.1% gelatin solution. Gelatin
did improve cell adhesion to tissue culture plastic (Figure 5d).
On gelatin-coated lms, cells appeared to grow in aggregates
and were detached from the center of the well during the PBS
rinse (Figure SI1). Because the detrimental eect was not
observed in the control, it may be due to interactions (or lack
thereof) between the chitosan surface and gelatin coating. At
day 1, there were no signicant dierences between cell
concentrations (Figure 5e). By day 5, the tissue culture plastic
conditions contained roughly twice the amount of adherent
viable cells as day 1. Cells grew on the chitosan lms to a lesser
extent but more importantly remained adherent and viable.
To translate the culture to 3D, chitosan sponges with aligned
microtubular pores were fabricated using a directional freezing
technique (Figure 6a). DrAMPCs were cultured in chitosan
sponges for 1 week (Figure 6b) or 2 weeks (Figure SI3). At
both time points, cells showed adhesion on all sponges (1, 2,
4% chitosan) and formed a nearly conuent monolayer. When
treated with 20-hydroxyecdysone, a small fraction (<1%) of
cells on the chitosan scaolds dierentiated (Figure 6c). The
average myocyte length on tissue culture plastic is 82 μm±40
μm(n= 19) and the average myocyte length on chitosan is 68
±34 μm(n= 7). The lengths are not statistically signicant
via unpaired ttest with an p-value of 0.5568. The longest
myocyte observed on plastic was 143 μm while the longest
myocyte observed on chitosan was 120 μm. Myocyte lengths
between chitosan scaolds did not dier signicantly. In select
areas of growth, cells appeared to be in alignment with the
length of the microtubular pores (Figure 6d). Although cells
grown on the 1% sponges exhibited strong adhesion, the
sponges were extremely fragile and did not retain an aligned
pore morphology when sectioned. After 2 weeks in culture, the
1% sponges disintegrated into sheet-like pieces while the 2%
and 4% sponges were more durable. The mechanical properties
of the sponges can be tuned via chitosan concentration.
Elastic modulus values of the 2% and 4% sponges were
determined via hydrated compression testing (Figure 6e). The
stiness of both sponges in the cross-section direction was
approximately 2 kPa. The moduli diered in the lateral
direction, the 4% sponge being approximately twice as stias
the 2% sponge.
Preliminary data on the thermal degradation of chitosan was
collected to provide initial insight into how chitosan sponges
degrade under simulated cooking. Thermogravimetric analysis
with ramped heat was performed from room temperature to
500 °C with a rate of 20 °C per minute. Chitosan powder
Figure 4. Conrmation of muscle identity and cellular contractions induced by extracellular potassium. (a) Confocal microscopy image of
DrAMPCs immunostained for ecdysone receptor and DAPI. (b) Fluorescence microscopy image of DrAMPCs immunostained for myosin heavy
chain and DAPI after 5 days of dierentiation with 1 μg/mL 20-HE in EC405. (c) Phase contrast image of DrAMPCs immediately before and after
treatment with media supplemented with 600 mM K+. Red circles indicate myoblasts contracting upon K+treatment.
ACS Biomaterials Science & Engineering Article
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begins to degrade at 325 °C and degradation is not
signicantly aected by heating rate (Figure SI2). There are
three phases of degradation: (1) volatilization of residual
materials, (2) chitosan chain degradation, and (3) decom-
position of remaining carbon.
Nutritional Properties of Insect vs Mammalian
Muscle Cell Cultures. In order to probe nutrition dierences
between mammalian and insect muscle cells, we measured
cellular protein and select mineral content of DrAMPC and
C2C12 cells. Both cell cultures were assayed for total protein,
iron and zinc (Table 1). Per cell, the C2C12 cells contain more
(by factors of 2.5, 2.9, and 3.4, respectively) protein, iron, and
zinc than DrAMPC cells. However, the C2C12 cells (average
diameter = 19.60 μm) are much larger than DrAMPCs
(average diameter = 5.72 μm). Correcting for cell size,
DrAMPCs contain a higher density of all three compounds per
unit volume. These results correlate with in vivo nutrient
values for the corresponding whole organisms as fruit y tissue
contains equivalent protein and a higher density of iron and
zinc compared to mouse tissue (Table SI4). This provides
preliminary evidence that as insects are often more nutrient
rich compared to mammalian tissue, insect cells may also be
more nutritious than mammalian cells.
In a simple attempt to increase iron content, we cultured
DrAMPCs in media supplemented with iron-fortied serum
(136 μM Fe) and compared the cellular iron content to
DrAMPCs cultured with media supplemented with conven-
tional serum (24 μM Fe). The iron-fortied DrAMPCs
contained twice the concentration of iron compared to the
control, although the fortied media contained roughly 6-fold
the amount of iron as the control media (Table 2). This
indicates that while media can be formulated to inuence
nutritional content, there is a limit to the amount of minerals
the cells can uptake.
Key challenges in the eld of cultured muscle biomass for
nonmedical applications can be addressed with invertebrate
cell sources. The main challenges include producing cells in a
cost-ecient manner and transforming the cell mass into
functional constructs. The majority of cell lines that have been
scaled for industrial production are derived from humans (e.g.,
HEK 293, HeLa S3, WI-38), rodents (e.g., CHO-K1, NS0,
BEK) and insects (e.g., S2, Sf9, High Five).
In most cases
these cells are not the end-product themselves, but rather are
utilized to produce valuable therapeutic proteins (e.g.,
antibodies, cytokines, growth factors). The most commonly
utilized cell lines share characteristics in that they are
immortalized and can achieve high growth densities in
serum-free, suspension culture.
So far, muscle cells have only been produced in relatively
small quantities for research. A key step toward industrial-scale
muscle cell culture is formulation of, and adaptation to, serum-
free media. Serum is expensive and susceptible to batch-to-
batch variability and cost uctuations based on supply
For cultured meat applications, serum must be
Figure 5. Adhesion and growth of insect muscle cells mushroom chitosan lms. (a) SEM images of the middle and edge morphology of a 2%
chitosan lm. Films of other concentrations were similar in appearance. (b) Fluorescence microscopy images of DrAMPCs at medium (75 000
cells/cm2) and high (300 000 cells/cm2) seeding densities on chitosan lms of variable concentrations after 24 h in culture. (c) Viable cells on
chitosan lms or control conditions quantied via MTS assay. Films and controls were assayed after 24 h in culture (initial seeding density of
300 000 cells/cm2) as they were (Total) or after being aspirated and rinsed with PBS (Adherent). (d) Percentage of adherent, viable cells
determined via MTS assay on untreated or 0.1% gelatin coated lms or controls. (e) Viable, adherent cells determined via MTS assay quantied
after 24 h and on day 5 of culture.
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Figure 6. Adhesion and dierentiation of insect muscle cells on chitosan sponge scaolds. (a) SEM images of cross-section and lateral views of a
2% chitosan sponge. Sponges of other concentrations were similar in appearance. (b) Confocal images of DrAMPCs on chitosan sponges of
variable concentrations after 1 week in culture in control media. (c) Fluorescence microscopy images of chitosan sponges with and without
hormone treatment. (d) Fluorescence microscopy image of DrAMPCs on a 4% chitosan sponge after 10 days of growth in EC405 media. The
white arrow indicates the orientation of the aligned microtubular pore. (e) Elastic moduli of chitosan sponges obtained via hydrated compression
tests performed in either the cross-sectional or lateral direction (error bars are standard deviations, n= 5). The schematic demonstrates the
direction of compression.
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eliminated from culture media to reduce costs and allow for
animal-free production. Serum-free media formulations have
been reported for mammalian muscle cells; however, they are
supplemented with animal-derived or recombinant proteins
(e.g., fetuin, broblast growth factor, insulin)
and, to our
knowledge, not commercially available. In this work, a D.
melanogaster muscle progenitor cell line was successfully
adapted to Ex-Cell 405 serum-free insect media. Although
this particular medium is proprietary, other insect cells have
been adapted from Ex-Cell 405 media to dened, low-cost
formulations with IPL-41 basal media (the preparation is
publicly available).
Future work could include optimizing
in-house media formulations.
The extended survival and proliferation of insect muscle cells
in the absence of fresh media is demonstrated in this work and
by previous studies from our lab.
We previously observed
that primary cultures of M. sexta cells survived over 75 days in
the same media and theorized the eect may be due to
supporting yolk and fat cell types which store nutrients. The
present work utilized an immortalized cell line and we
observed cell survival without nutrient exchange for more
than two months. We propose that the extended survival may
be attributed to dierences in mammalian versus insect
metabolism. In a study comparing mammalian and insect
glucose and glutamine metabolism, insect cells uniquely
channeled glucose metabolites into the tricarboxylic acid
cycle which produced more energy per unit glucose than
Insect cells also do not produce signicant lactate,
the accumulation of which signicantly lowers the pH of
mammalian muscle cell cultures.
Mammalian muscle cells generally grow as adherent rather
than suspension cultures. Cost-ecient production would
require suspension cultures of stem cells with myogenic-
lineage, adaptation of muscle progenitor cells to suspension
cultures or development of more complex bioreactor systems
suitable for adherent cell types (e.g., microcarriers, hollow
bers). The results from the present study demonstrated that
the DrAMPC line of invertebrate muscle cells can be grown in
single-cell suspension cultures and transition back to
monolayer cultures after expansion. Dextran sulfate supple-
mentation was sucient to reduce cell aggregation, and shaker
ask agitation kept cells in suspension. Removal of dextran
sulfate and reduction of cell density in static cultures promoted
monolayer culture and cell elongation. This versatility and
adaptability allows for streamlined production and scale-up.
For industrial bioprocesses, it will be advantageous to
control proliferation and dierentiation of muscle cells via
external factors. Methoprene and 20-hydroxyecdysone have
previously been shown to aect the growth and development
of insect cellsin vitro.
In the present study, methoprene
increased proliferation and 20-hydroxyecdysone triggered
dierentiation in both serum-supplemented and serum-free
cultures. Methoprene is used as an insecticide generated via
chemical synthesis
for the production of many agricultural
products. It poses minimal risk to the environment, and
guidelines for human consumption have been developed
(recommended exposure is set at 0.3750 mg/day for the
average adult).
The hormone 20-hydroxyecdysone is
produced by plants as well as insects and has been produced
in plant in vitro cultures.
It is also safe for human
consumption and even promoted as a tness supplement.
When applying this technology for food purposes, it is
important to consider potential hazards to human health.
Human health risks could arise from the original insect species,
the derived cell cultures, or the circulated media. Although
many insect species are edible and nutritious, some species
present risks in the form of antinutrient substances, allergens,
synthesized defense toxins, pesticides, and microbial patho-
gens. These risks can generally be avoided by selecting safe
species, decontamination and raising the insects on nontoxic
D. melanogaster ies are safe to eat and sold by
companies to eat as whole larvae, oils, or powder. Because D.
melanogaster are safe to consume, we assume cells cultured
from fruit ies would be safe to consume as well, given that the
cultures are fed with food-grade growth media. Human
pathogens have not been detected in insect cell cultures;
however, some cell lines can perform posttranslational
modications to proteins that may trigger human allergies.
Due to ExCell 405 being a proprietary media formulation, we
are unsure whether it contains any components that could be
dangerous for consumption. However, in-house serum-free
insect media formulations typically contain basal media, soy
protein extract, yeast extract, and lipids that are not toxic to
Cultured insect cells may be safer than whole
insects as the restricted number of cell types and a controlled
environment may eliminate the presence of certain allergens
and toxins. While primary cell lines are likely safe, concerns
may rise for continuous or immortalized cell lines. Sponta-
neously immortalized cell lines may develop mutations
overtime, and it is unclear whether this may pose a safety
risk. Genetically immortalized cells may present additional
concerns. The D. melanogaster adult muscle precursor-like cell
line used in this study was immortalized via the oncogenic
protein RasV12 and expresses GFP. Ingestion of GFP is
unlikely to be detrimental to human health. Farmed meat likely
contains cancerous tissue at times and this is probably not a
health risk as (1) cancer is not contagious and (2) proper
cooking and digestion processes should breakdown cancerous
cells. Similarly, dead cultured cells or tissues should not pose a
risk for human consumption. Regardless, it is important for
further research to take place surrounding the ingestion of
cultured and genetically modied cell-based foods.
Large, aligned muscle tissues are necessary to produce
bioactuators with useful contractile force and structured meat-
Table 1. Protein and Mineral Content, Cell Dimensions of Immortalized Mouse, and Fly Muscle Cell Lines
cell line protein (pg/cell) iron (pg/cell) zinc (pg/cell) suspended cell diameter (μm)
C2C12 1393 ±119 35.39 ±0.34 156.14 ±3.08 19.60 ±1.95
DrAMPC 558 ±67 12.09 ±0.40 46.02 ±2.16 5.72 ±0.81
Table 2. Iron Content of Bovine Serum and Insect Muscle
Cells Cultured in Media Supplemented with 10% Serum for
5 Days
iron content of
serum (μM) iron content of DrAMPCs cultured in
10% serum (μM)
fetal bovine
serum 24.35 ±0.58 27.06 ±0.90
serum 135.65 ±5.51 62.40 ±1.25
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like tissues for food. The construction of three-dimensional
and functional mammalian muscle tissues has been widely
Insect muscle constructs are remarkably less
researched and consist mainly of muscle explants or primary
cultures self-assembled in PDMS molds.
We evaluated
mushroom-derived chitosan as a scaolding biomaterial to
support in vitro culture of insect muscle cells. Chitosan was
selected as the biomaterial of choice for this system as it is (1)
a well-researched biomaterial in the eld of tissue engineering,
(2) edible, (3) inexpensive, (4) can be acquired from animal-
free sources (fungi, microbes), (5) is approved for human
consumption, (6) may supply health benets, and (7) scaold
techniques for three-dimensional muscle culture have been
In a feasible production scenario, insect cells
would rst be expanded in a single cell suspension culture and
subsequently seeded on a static scaold for tissue maturation.
Therefore, adhesion, viability, and dierentiation on cell-
scaold constructs are more important factors than growth.
Although the gelatin coating did not increase cell adhesion to
chitosan lms, a dierent outcome may result from creating a
chitosan-gelatin composite solution before casting the lm as
opposed to a gelatin coating. Other materials to investigate
include collagen and laminin, although it is important to
remember the design requirement of minimizing animal-
derived byproducts. Because cells can form a conuent layer on
nonfunctionalized chitosan lms and sponges, a coating
material may be unnecessary.
The purpose of modulating scaold stiness is to have some
control over the texture and palatability of the end-product.
One long-term goal of the research area is to have three-
dimensional cell-scaold constructs with mechanical properties
similar to meat. The elastic modulus of bovine muscle (mixed
ber orientation) is 1.5 kPa.
Chitosan sponges can be
fabricated to have a similar stiness, implying a chitosan-based
cultured meat product could have similar texture to familiar
meat products. It will also be important to investigate the
mechanical properties of seeded scaolds and the eect of
cooking temperature and time.
The most prevalent challenge in this study was promoting
homogeneous dierentiation, as the majority of cells retained a
spherical morphology indicative of their proliferative state.
However, on plastic and chitosan scaolds, myosin heavy
chain-expressing cells could be generated (albeit at low
eciency) after treatment with 20-hydroxyecdysone. In all
control samples (plastic and sponges not treated with 20-
hydroxyecdyone), no myosin heavy chain-expressing cells were
observed. This supports the conclusion that while DrAMPCs
have the capacity to dierentiate to a muscle phenotype on
plastic and 2D and 3D chitosan scaolds, the system is lacking
an element or elements necessary for robust dierentiation.
There is evidence from previous studies that insect muscle cells
may require supporting cell types, specically neurons, in order
to fully dierentiate into mature myotubes.
Our future
work will focus on achieving a greater percentage of
dierentiated cells by implementing co-culture and electrical
stimulus methods.
This work lays the foundation for insect muscle tissue
engineering as a tool for cultured meat production and
bioactuation. While previous research has demonstrated insect
cell types purposed for protein production can be adapted to
serum-free medium and cultured in suspension, it was
previously unclear whether the same benets applied to insect
muscle cells. There was also a gap in scaold design for insect-
based constructs, as scaolds have primarily been composed of
PDMS, which is inapplicable to food-related technologies with
these in vitro tissues. Furthermore, while edible insects are
lauded for their nutrient density, it was unknown whether in
vitro insect cells have a desirable nutrition prole. To address
these issues, we used genetically immortalized and highly
proliferative D. melanogaster adult muscle progenitor-like cells
to explore the feasibility of scalable cell production, the
integration of scaold techniques and the evaluation of
nutritional content. We successfully adapted our cells to an
inexpensive, serum-free media formulation and transitioned the
cells from adherent to suspension cultures. We also compared
the long-term survival of DrAMPCs in limited nutrient
conditions to survival of the mouse-derived myoblast C2C12
cell line to highlight the benets of the insect cells. To develop
scaolds inspired by the insect cuticle, we fabricated lms and
sponges from mushroom-derived chitosan to develop 2D and
3D culture systems. Lastly, we analyzed the protein, iron, and
zinc content of the DrAMPC cells compared to C2C12 cells to
show the nutritional value of these in vitro insect cell cultures.
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsbiomater-
Details about the adaptation of the cell line to serum-
free media, antibody information, and detailed descrip-
tion of statistical analysis methods; additional gures
include microscopy images from gelatin coating experi-
ments, DrAMPC growth on chitosan sponges at 2 weeks
in culture, and DrAMPCs in long-term starvation culture
as well as data from thermogravimetric analysis of
chitosan (PDF)
Corresponding Author
David L. Kaplan: 0000-0002-9245-7774
The authors declare no competing nancial interest.
We thank the NSF (IOS-1557672) for support. We would like
to acknowledge Chinova Bioworks for donating mushroom
chitosan for scaold fabrication. Additional thanks to New
Harvest for nancial and scientic support. This work was
supported by the NIH Research Infrastructure grant NIH S10
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ACS Biomaterials Science & Engineering Article
DOI: 10.1021/acsbiomaterials.8b01261
ACS Biomater. Sci. Eng. XXXX, XXX, XXXXXX
... In the past 5 years, research focused on cell-based meats has accelerated from the tasting of the cell-cultured hamburger in 2013 (Zaraska, 2013) and early publications, including lifecycle assessments and basic research (Tuomisto and Teixeira de Mattos, 2011;Post, 2012Post, , 2014, to a growing start-up community, updated life cycle assessments, and publications focused on refining the technologies required to accelerate cellbased meat production (Tuomisto et al., 2014;Krieger et al., 2018;Rubio et al., 2019). To date, research decisions in cell-based meat production, such as selection of cell species and cell type have been largely driven by market size and environmental impact (Rodriguez-Fernandez, 2019), rather than suitability of cells species and types suitable for large scale bioreactor cultivation. ...
... Many scaffolds that support various cell cultures use chitosan solutions as low as 0.5% (Katalinich, 2001). To create films to support cell culture, chitosan solutions are cast on glass substrates and allowed to dry, then rinsed with buffer to neutralize the acetyl groups, washed with water or PBS and sterilized with 70% ethanol and UV light prior to cell seeding (Rubio et al., 2019). Chitosan can also be manipulated to produce physically associated or cross-linked hydrogels (Drury and Mooney, 2003), porous sponges with tunable mechanical properties and pore size distributions (Jana et al., 2013;Rubio et al., 2019) and dry or wet spun fibers (Croisier and Jérôme, 2013). ...
... To create films to support cell culture, chitosan solutions are cast on glass substrates and allowed to dry, then rinsed with buffer to neutralize the acetyl groups, washed with water or PBS and sterilized with 70% ethanol and UV light prior to cell seeding (Rubio et al., 2019). Chitosan can also be manipulated to produce physically associated or cross-linked hydrogels (Drury and Mooney, 2003), porous sponges with tunable mechanical properties and pore size distributions (Jana et al., 2013;Rubio et al., 2019) and dry or wet spun fibers (Croisier and Jérôme, 2013). ...
... Earthworm muscle, shown in Figure 4d, has been evaluated for controllable drug delivery by Tanaka's group [116]. The natural combined mechanism of the longitudinal and circular actuations renders a more favorable laminate geometry compared to skeletal muscles [121][122][123]. Their pump achieved a flow rate of 5.0 µL s −1 , which is about 3-4 orders higher than a similar form based on a cardiomyocyte pump [124,125]. ...
... Although this light-based micromachine may not be used in the body at present, this work highlights the concept of potential responsive microsystems. Insect muscles are among the toughest natural actuators on earth as they can tolerate a wider range of external and internal conditions than birds, mammals, and vertebrate ectotherms [122,160]. One pair of micro-tweezers based on insect muscles was developed by Akiyama et al. [71]. ...
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The choice of actuators dictates how an implantable biomedical device moves. Specifically, the concept of implantable robots consists of the three pillars: actuators, sensors, and powering. Robotic devices that require active motion are driven by a biocompatible actuator. Depending on the actuating mechanism, different types of actuators vary remarkably in strain/stress output, frequency, power consumption, and durability. Most reviews to date focus on specific type of actuating mechanism (electric, photonic, electrothermal, etc.) for biomedical applications. With a rapidly expanding library of novel actuators, however, the granular boundaries between subcategories turns the selection of actuators a laborious task, which can be particularly time-consuming to those unfamiliar with actuation. To offer a broad view, this study (1) showcases the recent advances in various types of actuating technologies that can be potentially implemented in vivo, (2) outlines technical advantages and the limitations of each type, and (3) provides use-specific suggestions on actuator choice for applications such as drug delivery, cardiovascular, and endoscopy implants.
... As such, these materials are promising options for cultivated meat production. Naturally derived polysaccharides: alginate, chitosan and konjac, have also been successfully explored for cultivated meat and tissue engineering applications [25][26][27][28]. CNF has inert, non-toxic, biodegradable properties, and is obtained from renewable sources (e. ...
... The acetyl groups on konjac molecules were deacetylated in alkaline conditions and formed hydrogen-bonded networks at high temperature [49] during steam sterilization. The structure of chitosan films were stabilized through neutralization with alkaline solution [27]. ...
Biomaterial scaffolds are critical components in cultivated meat production for enabling cell adhesion, proliferation, differentiation and orientation. Currently, there is limited information on the fabrication of edible/biodegradable scaffolds for cultivated meat applications. In the present work, several abundant, naturally derived biomaterials (gelatin, soy, glutenin, zein, cellulose, alginate, konjac, chitosan) were formed into films without toxic cross-linking or stabilizing agents. These films were investigated for support of the adhesion, proliferation and differentiation of murine and bovine myoblasts. These biomaterials supported cell viability, and the protein-based films showed better cell adhesion than the polysaccharide-based films. Surface patterns induced cell alignment and guided myoblast differentiation and organization on the glutenin and zein films. The mechanical properties of the protein films were also assessed and suggested that a range of properties can be achieved to meet food-related goals. Overall, based on adherence, proliferation, differentiation, mechanics, and material availability, protein-based films, particularly glutenin and zein, showed the most promise for cultivated meat applications. Ultimately, this work presents a comparison of suitable biomaterials for cultivated meat applications and suggests future efforts to optimize scaffolds for efficacy and cost.
... This discordance occurs because myoglobin is repressed by cultured cells in the presence of oxygen, and because commonly used culture media such as IMDM, RPMI1640, and DMEM contain minimal iron content. This problem can be addressed if the media is supplemented with iron, but this supplementation remains limited [41][42][43]. A further problem relates to taste. ...
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The constant growth of the population has pushed researchers to find novel protein sources. A possible solution to this problem has been found in cellular agriculture, specifically in the production of cultured meat. In the following review, the key steps for the production of in vitro meat are identified, as well as the most important challenges. The main biological and technical approaches are taken into account and discussed, such as the choice of animal, animal-free alternatives to fetal bovine serum (FBS), cell biomaterial interactions, and the implementation of scalable and sustainable biofabrication and culturing systems. In the light of the findings, as promising as cultured meat production is, most of the discussed challenges are in an initial stage. Hence, research must overcome these challenges to ensure efficient large-scale production.
... Stephens et al. 2018;Rubio et al. 2019;Tuomisto 2019; Post et al. 2020). Only a few studies to date have quantified the GHG emissions of microbial proteins or cultured meat, suggesting GHG emissions at the level of poultry meat(Tuomisto and Teixeira de Mattos 2011;Mattick et al. 2015;Souza Filho et al. 2019;Tuomisto 2019). ...
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Cultivated meat, also known as cultured or cell-based meat, is meat produced directly from cultured animal cells rather than from a whole animal. Cultivated meat and seafood have been proposed as a means of mitigating the substantial harms associated with current production methods, including damage to the environment, antibiotic resistance, food security challenges, poor animal welfare, and—in the case of seafood—overfishing and ecological damage associated with fishing and aquaculture. Because biomedical tissue engineering research, from which cultivated meat draws a great deal of inspiration, has thus far been conducted almost exclusively in mammals, cultivated seafood suffers from a lack of established protocols for producing complex tissues in vitro. At the same time, fish such as the zebrafish Danio rerio have been widely used as model organisms in developmental biology. Therefore, many of the mechanisms and signaling pathways involved in the formation of muscle, fat, and other relevant tissue are relatively well understood for this species. The same processes are understood to a lesser degree in aquatic invertebrates. This review discusses the differentiation and maturation of meat-relevant cell types in aquatic species and makes recommendations for future research aimed at recapitulating these processes to produce cultivated fish and shellfish.
Cell-cultured fat could provide important elements of flavor, nutrition, and texture to enhance the quality and therefore expand consumer adoption of alternative meat products. In contrast to cells from livestock animals, insect cells have been proposed as a relatively low-cost and scalable platform for tissue engineering and muscle cell-derived cultured meat production. Furthermore, insect fat cells have long been cultured and characterized for basic biology and recombinant protein production but not for food production. To develop a food-relevant approach to insect fat cell cultivation and tissue engineering, Manduca sexta cells were cultured and induced to accumulate lipids in 2D and 3D formats within decellularized mycelium scaffolding. The resultant in vitro fat tissues were characterized and compared to in vivo fat tissue data by imaging, lipidomics, and texture analyses. The cells exhibited robust lipid accumulation when treated with a 0.1 mM soybean oil emulsion and had "healthier" fat profiles, as measured by the ratio of unsaturated to saturated fatty acids. Mycelium scaffolding provided a simple, food-grade approach to support the 3D cell cultures and lipid accumulation. This approach provides a low-cost, scalable, and nutritious method for cultured fat production.
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This review article highlights recent advances in designing biomaterials to be interfaced with food and plants, with the goal of enhancing the resilience of the AgroFood infrastructure by boosting crop production, mitigating environmental impact, and reducing losses along the supply chain. Special attention is given to innovations in biomaterial‐based approaches and platforms for 1) seed enhancement through encapsulation, preservation, and controlled release of payloads (e.g., plant growth‐promoting microbes) to the seeds and their rhizosphere; 2) precision delivery of multi‐scale payloads to targeted plant tissues, organelles, and vasculature; 3) edible food coatings that regulate gas exchanges and provide antimicrobial properties to extend the shelf life of perishable food; and 4) food spoilage detection based on different sensor/reporter systems. Within each domain, biomaterials design principles, emerging micro‐/nanofabrication strategies, and the advantages and disadvantages of different delivery/preservation/sensing platforms are introduced and critically discussed. Views of future requirements, aims, and trends are also given based on the opportunities and challenges of applying biomaterials in the AgroFood system.
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Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high‐quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties. Cultivating meat from cells is a promising solution to the environmental, ethical, health, and food‐security challenges associated with conventional meat production. Cultivated meat will require the development of scalable, low‐cost, and edible or biodegradable scaffolds to support cell growth. This review discusses the unique challenges of cultivated meat scaffolding and highlights promising materials and processing methods worthy of further investigation.
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Actuation is essential for artificial machines to interact with their surrounding environment and to accomplish the functions for which they are designed. Over the past few decades, there has been considerable progress in developing new actuation technologies. However, controlled motion still represents a considerable bottleneck for many applications and hampers the development of advanced robots, especially at small length scales. Nature has solved this problem using molecular motors that, through living cells, are assembled into multiscale ensembles with integrated control systems. These systems can scale force production from piconewtons up to kilonewtons. By leveraging the performance of living cells and tissues and directly interfacing them with artificial components, it should be possible to exploit the intricacy and metabolic efficiency of biological actuation within artificial machines. We provide a survey of important advances in this biohybrid actuation paradigm.
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For several tissue engineering applications, in particular food products, scaling up culture of mammalian cells is a necessary task. The prevailing method for large scale cell culture is the stirred tank bioreactor where anchor dependent cells are grown on microcarriers suspended in medium. We use a spinner flask system with cells grown on microcarriers to optimize the growth of bovine myoblasts. Freshly isolated primary cells were seeded on microcarriers (Synthemax(®), CellBIND(®) and Cytodex(®) 1 MCs). In this study, we provide proof of principle that bovine myoblasts can be cultured on microcarriers. No major differences were observed between the three tested microcarriers, except that sparsely populated beads were more common with CellBIND(®) and Synthemax(®) II beads suggesting a slower initiation of exponential growth than on Cytodex(®). We also provide direct evidence that bovine myoblasts display bead-to-bead transfer. A remarkable pick up of growth was observed by adding new MCs. Bovine myoblasts seem to behave like human mesenchymal stem cells. Thus, our results provide valuable data to further develop and scale-up the production of bovine myoblasts as a prerequisite for efficient and cost-effective development of cultured meat. Applicability to other anchorage dependent cells can extend the importance of these results to cell culture for medical tissue engineering or cell therapy.
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Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass.
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Some pesticides are applied directly to aquatic systems to reduce numbers of mosquito larvae (larvicides) and thereby reduce transmission of pathogens that mosquitoes vector to humans and wildlife. Sustained, environmentally-safe control of larval mosquitoes is particularly needed for highly productive waters (e.g., catchment basins, water treatment facilities, septic systems), but also for other habitats to maintain control and reduce inspection costs. Common biorational pesticides include the insect juvenile hormone mimic methoprene and pesticides derived from the bacteria Bacillus thuringiensis israelensis, Lysinibacillus sphaericus and Saccharopolyspora spinosa (spinosad). Health agencies, the public and environmental groups have especially debated the use of methoprene because some studies have shown toxic effects on non-target organisms. However, many studies have demonstrated its apparent environmental safety. This review critically evaluates studies pertinent to the environmental safety of using methoprene to control mosquito larvae, and provides concise assessments of the bacterial larvicides that provide sustained control of mosquitoes. The review first outlines the ecological and health effects of mosquitoes, and distinguishes between laboratory toxicity and environmental effects. The article then interprets non-target toxicity findings in light of measured environmental concentrations of methoprene (as used in mosquito control) and field studies of its non-target effects. The final section evaluates information on newer formulations of bacterially-derived pesticides for sustained mosquito control. Results show that realized environmental concentrations of methoprene were usually 2–5 µg/kg (range 2–45 µg/kg) and that its motility is limited. These levels were not toxic to the vast majority of vertebrates and invertebrates tested in laboratories, except for a few species of zooplankton, larval stages of some other crustaceans, and small Diptera. Studies in natural habitats have not documented population reductions except in small Diptera. Bacterial larvicides showed good results for sustained control with similarly limited environmental effects, except for spinosad, which had broader effects on insects in mesocosms and temporary pools. These findings should be useful to a variety of stakeholders in informing decisions on larvicide use to protect public and environmental health in a ‘One Health’ framework.
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The pig is recognized as a valuable model in biomedical research in addition to its agricultural importance. Here we describe a means for generating skeletal muscle efficiently from porcine induced pluripotent stem cells (piPSC) in vitro thereby providing a versatile platform for applications ranging from regenerative biology to the ex vivo cultivation of meat. The GSK3B inhibitor, CHIR99021 was employed to suppress apoptosis, elicit WNT signaling events and drive naïve-type piPSC along the mesoderm lineage, and, in combination with the DNA methylation inhibitor 5-aza-cytidine, to activate an early skeletal muscle transcription program. Terminal differentiation was then induced by activation of an ectopically expressed MYOD1. Myotubes, characterized by myofibril development and both spontaneous and stimuli-elicited excitation-contraction coupling cycles appeared within 11 days. Efficient lineage-specific differentiation was confirmed by uniform NCAM1 and myosin heavy chain expression. These results provide an approach for generating skeletal muscle that is potentially applicable to other pluripotent cell lines and to generating other forms of muscle.
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Ecdysteroids are secondary metabolites, widely distributed in the animal and plant kingdoms. They have a wide range of pharmacological effects in vertebrates, including mammals, most of which are beneficial for humans. Therefore, they have become compounds of interest for the pharmaceutical industry due to their adaptogenic, anabolic, hypoglycaemic, hypocholesterolaemic and antimicrobial activities, which are still being researched. Nowadays, ecdysteroids are present as active ingredients in bodybuilding supplements. Because of their complex structures, their chemical synthesis seems unprofitable and impractical. Due to high content of ecdysteroids in many plants, they are primarily obtained by extraction of the plant material. Plant in vitro cultures provide an alternative source of these compounds, helping to avoid problems associated with field production—such as variable yield or dependence on environmental factors, as well as limited availability of natural resources. Plant cell and tissue cultures may be suggested as alternatives for the production of plant biomass rich in pharmaceutically active ecdysteroids. Moreover, the use of common biotechnological strategies, such as elicitation or precursor feeding, may further increase the yield and improve production of these compounds. In this paper, we describe general information about ecdysteroids: their structure, biosynthesis, distribution, role in plants, and we review recent studies on micropropagation of ecdysteroid-producing plants and cell cultures, and potential ability of ecdysteroids enhancement in in vitro cultures.
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The development of serum-free and chemically defined media remains an important mission in the in vitro research of cells. The present study describes an improved serum-free medium that supports the proliferation and differentiation of chick embryonic primary skeletal myogenic cells, a cell type which has long been utilized for the study of cell differentiation. We show that serum can be replaced with transferrin, insulin, serum albumin, and fibroblast growth factor-2 as supplements to Dulbecco's modified Eagle minimum essential medium, with no loss in the ability of the medium to support proliferation and differentiation of myogenic cells. This medium has several additional advantages over serum-supplemented medium in that it also suppresses the proliferation of contaminating fibroblasts, and may allow the sensitive evaluation of the effects of various humoral factors on myogenic cells. We believe this medium will prove useful to the primary culture of chick myogenic cells.
Biological materials can adaptively respond to their environment, motivating their use as functional components of engineered machines. In article number 1700030, Rashid Bashir and co-workers present a skeletal muscle-powered bio-hybrid robot (bio-bot) that can adapt to loss-of-function damage stimuli, completely healing and recovering functionality within two days. This presents a significant step forward in developing robust, resilient, and dynamically responsive biohybrid machines.
A deeper understanding of biological materials and the design principles that govern them, combined with the enabling technology of 3D printing, has given rise to the idea of "building with biology." Using these materials and tools, bio-hybrid robots or bio-bots, which adaptively sense and respond to their environment, can be manufactured. Skeletal muscle bioactuators are developed to power these bio-bots, and an approach is presented to make them dynamically responsive to changing environmental loads and robustly resilient to induced damage. Specifically, since the predominant cause of skeletal muscle loss of function is mechanical damage, the underlying mechanisms of damage are investigated in vitro, and an in vivo inspired healing strategy is developed to counteract this damage. The protocol that is developed yields complete recovery of healthy tissue functionality within two days of damage, setting the stage for a more robust, resilient, and adaptive bioactuator technology than previously demonstrated. Understanding and exploiting the adaptive response behaviors inherent within biological systems in this manner is a crucial step forward in designing bio-hybrid machines that are broadly applicable to grand engineering challenges.
A process is developed to engineer hydrogel microfibers with precise micropatterning of ECM protein adhesive sites in order to control the adhesion, spreading, and alignment of muscle cells. Uniquely, the ribbon-like microfibers wrap around the cells, aiding in uniaxial alignment and enabling the formation of multifiber bundles to form larger anisotropic skeletal muscle tissue.