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OPTIMIZATION OF BOVINE SATELLITE CELLS PROLIFERATION ON MICROCARRIERS

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  • New Harvest

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Using Synthemax and Cytodex micro carriers to grow bovine satellite cells in spinner flask suspension culture
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OPTIMIZATION OF BOVINE SATELLITE CELL PROLIFERATION ON
MICROCARRIERS
Daan Luining
2015
Supervisors:
prof. dr. Mark Post, Anon van Essen, Sanne Verbruggen,
Department of Physiology, Maastricht University
Examiner:
Dr. Arthur F.J. Ram, Department of Molecular Microbiology & Biotechnology
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Daan Luining
Noordeinde 52
2311CE Leiden
0653168063
Daan_luining101@hotmail.com
Prof. Dr. Mark J. Post
Universiteitssingel 50
6229 ER Maastricht
31-43-3881200
m.post@maatrichtuniversity.nl
Dr. Arthur F.J. Ram
Sylviusweg 72
2333 BE Leiden
31-71-527 4914
a.f.j.ram@biology.leidenuniv.nl
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Contents
Abstract ................................................................................................................................................... 3
Introduction ............................................................................................................................................. 4
Background .......................................................................................................................................... 4
Aim: ..................................................................................................................................................... 8
Material and Methods. ............................................................................................................................ 9
Cell culture........................................................................................................................................... 9
Micro carriers ...................................................................................................................................... 9
Micro carrier cell culture in spinner-flasks .......................................................................................... 9
Qubit™ dsDNA assay ......................................................................................................................... 10
MTS assay .......................................................................................................................................... 10
Hoechst staining ................................................................................................................................ 10
Rhodamin staining ............................................................................................................................. 11
EDTA detachment .............................................................................................................................. 11
RNA isolation and Q-PCR ................................................................................................................... 11
Statistics ............................................................................................................................................ 11
Results ................................................................................................................................................... 12
Cell inoculation density on micro carrier experiment ....................................................................... 12
Cell inoculation density experiment with extended growth ............................................................. 14
Detachment of cells from Synthemax coating (2D) using Ethylenediaminetetra acetic acid (EDTA) 16
Bead-to-bead transfer experiment ................................................................................................... 18
Micro carrier variation experiment ................................................................................................... 20
Discussion .......................................................................................................................................... 22
Conclusion ............................................................................................................................................. 24
Future Prospect ..................................................................................................................................... 25
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Abstract
Using Satellite cells for in vitro meat production is an alternative for using life stock to meet the ever
increasing demand for meat. However, the method used to make the first laboratory grown
hamburger is not suitable for large scale production and should be optimized before it can compete
with conventional meat production. Using micro carriers decreases the volume that is needed to
grow large amounts of cells. The micro carriers provide the cells with a surface to bind and
proliferate on. The cell-bead suspension was cultured in small 125ml spinner flasks to keep the cells
and beads in suspension. The Qubit™ dsDNA analyses was chosen for measuring the growth of the
cells during culturing. When Synthemax and Cellbind beads were used, an optimum was found of 3
million cells per 183cm2 of beads to colonize all the beads with at least 1 cell per bead. When
Cytodex1 beads were used only 1 million cells were needed to colonize 183cm2 of beads. When
proliferation efficiency was compared, Cytodex1 beads showed the highest DNA concentration
compared to Cellbind and Synthemax. Bead to bead transfer was achieved by overnight intermittent
stirring. This was performed using the Synthemax and the Cytodex1 beads. To detach cells from the
beads, an experiment was performed using EDTA to destabilize anchoring proteins. This method
cannot be used for cells detachment since none of the detached cells survived the transfer. These are
small steps towards a sustainable meat industry. Still, there are many hurdles to overcome before
this technique is ready to compete with the current methods.
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Introduction
Background
We shall escape the absurdity of growing a whole chicken in order to eat the breast or wing, by
growing these parts separately under a suitable medium.
Winston Churchill, Fifty Years Hence, The Strand Magazine (December 1931)
Sir Winston Churchill foresaw in 1931 the inefficiency of growing an entire animal just to consume
parts of it. Nearly twenty years later the first ideas for culturing tissue for consumption were
proposed by Willem van Eelen. In 1999 he patented his idea about in vitro meat (Van Eelen et al.
1999). In 2011 Professor of Vascular Physiology and Tissue Engineering at Maastricht University,
Mark Post started a proof of principle project to produce the world's first laboratory grown
hamburger made from cultured muscle cells. Two years later on the 5th of August 2013, this project
was successfully completed by presenting the hamburger during a TV-talk show at which it was
cooked and eaten in front of a live audience and was broadcasted worldwide. The cost of this success
was a staggering €250.000. To make this technology viable and cost effective, optimization and
scaling experiments are necessary.
For the production of in vitro meat, bovine satellite cells are used to form bio artificial muscles
(BAM). In the body, these cells are responsible for regeneration of muscle tissue when cells get
damaged or for muscle growth (Collins et al. 2005). When observed under a microscope the cells are
mononuclear and can be found between the basal membrane and the sarcolemma of nearby muscle
fibers in skeletal muscles in mammals (Mauro 1961). Myosatellites can both self-renew by
proliferation and grow out to myofibers by differentiation (Yin et al. 2013). CD34, Myf5 and β-
galactosidase are expressed in different stages of satellite cell activation proliferation and
differentiation. These genes are used as genetic markers for satellite cells during maturation
(Beauchamp et al. 2000). The development and gene expression is shown in figure 1.
Figure 1. Visualization of satellite cell maturation from quiescent state into myofibers. Different genes are expressed during
the maturation process. These genes can be used as biomarkers connected to each maturation state.
(Zammit, Partridge et al. 2006)
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In order to make meat from satellite cells a few phases have been defined (fig 2). Satellite cells are
isolated from bovine skeletal muscle (Dodson et al. 1987); however, these cells are few in number
and decrease during aging (Schultz & Lipton 1982). Progress in the isolation of these cells has been
made, and due to the highly proliferative capability of these cells, only a few cells are needed to start
a cell culture. There are many methods to isolate satellite cells ranging from attachment speed
separation to magnetic bead separation and enzymatic digestion (Shefer & Zipora Yablonka-Reuveni
2005). Most have proven to work well.
When the cells have been isolated they are expanded in culture flasks with the appropriate medium.
This medium contains high amounts of animal serum, which makes it one of the bottlenecks of the
scaling process. Ongoing research is being performed to remove the dependence on animal derived
serum (van der Valk et al. 2010). During a 7 to 8 week period of culturing, the cells divide in up to 30
population doublings; a decrease in proliferation of cells cultured in vitro has previously been
reported (Machida et al. 2004), but theoretically these cells could reach 50 to 70 doublings.
After obtaining enough cells, they are split in portions of 1.5 million cells. Each portion is immersed in
a collagen/Matrigel gel in a culture dish. The cells are positioned around a central column of agarose
gel. This way the cells will organize into a circle shaped fiber with a diameter of 1 millimeter around
the agarose column. After 3 weeks the muscle fiber is harvested. For making an 85 gram hamburger
10 000 muscle strips were used (Post 2013). In total, the process from isolation till complete
hamburger takes around 3 months, which is shorter than raising an animal. However, the manual
labor and production cost still far outweigh the benefits of this technique.
Figure 2. Schematic workflow of the principle of in vitro skeletal muscle tissue culturing.
Therefore, new methods are being developed to reduce labor and production costs. One of these
methods involves growing the satellite cells in a 3D fermenter. A challenge in this method is that
satellite cells are surface dependent. Without a surface serving as the extracellular matrix, satellite
cells will undergo apoptosis (Frisch & Francis 1994). A solution to this problem is the use of micro
carriers. Micro carriers are spheres, or so-called beads, typically 100-200 μm in diameter, to which
cells can adhere and proliferate.
Cells can adhere to a wide variety of bead materials: glass, various plastics, metals, dextran, cellulose,
polylysine, collagen, gelatin and numerous extracellular matrix proteins. Plastics used in cell culture
include polystyrene, polyethylene, polycarbonate, Perspex™, PVC, Teflon™, cellophane, and cellulose
acetate. These organic materials are made by oxidation, strong acids, high voltage, UV light and as a
result, are negatively charged. Cells that grow attached on plastic surfaces should also attach and
grow on carriers.
Cell adhesion to beads is a multistep process involving initial contact of the cell with the surface, cell
spreading on the surface, and cell growth or differentiation (fig 3). Anchorage-dependent cells need a
certain amount of time to attach to these surfaces. This can be improved by coating the surfaces with
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proteins such as collagen or fibronectin. However, these substances are often derived from animal
products and are therefore not a suitable option for this particular study. A solution to this problem
could be the use of recombinant proteins produced in prokaryotic or lower eukaryotic cells.
However, disadvantages of this method are the high price of such products and the fact that media
for bacteria or yeast may contain some sort of animal-derived products. Because of these reasons,
micro carriers without an animal derived coating were used.
Figure 3. Schematic drawing of the attachment of cells to the micro carrier in 4 phases.
The advantage of using micro carriers in cell culture is that the volume that is needed to grow a large
amount of cells drastically decreases, and that cell production is scalable. When compared with other
monolayer or suspension techniques, stirred micro carrier cultures yield up to 100-fold as many cells
for a given volume of medium (GE Healthcare 2005). A downside of this method is that cells seem to
form aggregates and beads stick together, which may cause the cells to cease proliferating (Molnar
et al. 1997).
To realize mass production of satellite cells, a labor efficient system must be developed. The goal is
to optimize the large-scale cell production system to generate a large amount of cells in a short time
with the smallest possible amount of resources (culture medium, cells, and energy) and minimal
handling. For the very large-scale cultivation of stem cells for cultured beef, suspension cultures in
bioreactors are required (Tuomisto & Teixeira de Mattos 2011). To reach the high cell density,
different parameters need to be investigated and optimized. To keep costs low for the initial
experiments, 125 mL spinner flasks were used.
Initial cell-to-bead ratio is one of these parameters. When too few cells are used to colonize the
empty beads, cells may stop proliferating due to lack of cell-cell contact (Mather, Roberts 1998).
Studies have shown increased attachment rate with higher cell inoculation densities but higher
expansion with lower cell inoculation densities (Ng et al. 1996). Another obstacle to reach high cell
density is the transfer of cells to empty or newly added beads. Therefore a fed-batch system is
proposed where new beads are added to the culture when the cells reach near confluence. Agitated
stirring brings fresh beads in close contact with beads containing cells, thus enabling cell transfer
(Wang 1999). This way, when using a bioreactor, only fresh medium and beads can be added to a
closed system thus minimizing the risk of infection, aggregate formation (Ferrari 2012) and handling.
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During the culturing period, the growth of the cells will be monitored. This is a hurdle by itself
because taking a representable sample, pipetting the same amount of beads, and using an assay that
is compatible with the micro carriers turned out to be more of a challenge than previously was
expected. Here we compare two assays: the MTS assay, where a yellow tetrazole is reduced to
purple formazan in living cells, which absorbs light at 490-500 nm (Mosmann 1983) and the Qubit™
dsDNA assay which measures double stranded DNA after cells are lysed using a lysis buffer, and
signal is produced by adding a fluorochrome to the cell lysate that binds to the DNA.
In the final stage of culturing, cells need to be separated from the beads in order to enter the
differentiation and tissue formation stage. The beads are made of polystyrene and are inedible but
could be used multiple times. A difficulty we encountered was that the cells are strongly adherent to
the beads, and even with the use of trypsin, proved to be difficult to detach. Here we experimented
with a titer of Ethylenediaminetetra acetic acid (EDTA) to detach the cells from the beads. To
destabilize the cell-adhesion proteins, EDTA is added to the cell culture and chelates Ca2+ and Mg2+.
Selectins, integrins, cadherins, and Ig superfamily members (e.g iCAM, N-CAM) are proteins involved
in cell-surface adhesion (Hirano 1987). These proteins are dependent on ion flow of Ca2+ or Mg2+ (GE
Healthcare 2005), and therefore, the hypothesis is that this way cells can be detached from the micro
carriers without using trypsin.
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Aim:
The main objective of this study is to produce low cost, lab cultured beef for human consumption. A
proof of principle pilot study has been performed with success. The next step in this process is to
optimize, upscale, and reduce time and cost of production. Here we focus on using micro carriers in a
stirred tank fermenter to scale up the production of satellite cells.
Questions to be answered are:
What assay could be used for measuring cell concentration?
What is the optimal inoculation density of cells to micro carrier ratio for optimal growth?
Is bead to bead transfer possible during culturing?
What type of bead is most suitable for cell growth and expansion?
How can cells be retrieved from micro carriers?
By testing these parameters, we hope to achieve a reliable method to track satellite cell growth and
to optimize growth conditions for large-scale cell production. By measuring metabolic activity and
DNA concentration, we expect to track cell proliferation over time. Measuring cell density is a
challenge because the micro carriers don’t allow for visual confirmation by microscopy or
conventional counting methods like Trypan blue exclusion test of cell viability. Measuring DNA
content and metabolic activity doesn’t give an absolute cell number, but it allows tracking of cell
proliferation during culturing.
Next, we are testing different concentrations of cells to inoculate the micro carriers to determine the
optimal amount of cells that are needed to colonize all beads. When the beads are (near) confluence
more beads will be added to the culture, thus more surface is available for cells to support continued
growth. Also different types of micro carriers are available. Three different types will be tested for
their capability to bind cells, grow them over time, and bead to bead transfer abilities. In the final
stage of culturing the satellite cells need to be harvested from the beads since these are made of
polystyrene, which is inedible. Previous test have shown that the cells bind strongly to the bead
surface and are hard to detach without damaging or killing them.
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Material and Methods.
Cell culture
Growth medium used in all experiments was Dulbecco’s modified Eagle’s medium, advanced DMEM
(Gibco) supplemented with 20% heat inactivated fetal bovine serum, 10% horse serum, 5mM L-
glutamine, 1% Penicillin/Streptomycin/Fungizone. All media supplements were bought from
Invitrogen unless otherwise stated.
Culture flasks were coated with Matrigel™ (BD Bioscience, 356234) by diluting Matrigel™ in growth
medium 1:200, adding to culture flasks, and incubating for 1 hr at 37oC, 5% CO2. Cells were passaged
by aspirating the medium in the flask and washing the cell surface with PBS. Preheated 0,05%
trypsin/EDTA 1X (Gibco) was added to the flask and was incubated for 5 minutes in 37oC for cell
detachment from the surface. This was confirmed by microscopy. For trypsin inactivation, Trypsin
neutralizer was added in a 1:1 ratio. The cell suspension was transferred to a 50mL Falcon tube and
was centrifuged for 5 minutes at 1500 rpm. After centrifugation, the pellet was resuspended in
growth medium.
Cell counting was done by the Trypan blue exclusion method. Cells were stained with Trypan blue
and counted in a Countess® automated cell counter (Invitrogen, USA) by mixing 10 μL 0.4% Trypan
blue (Invitrogen) and 10 μL cell suspension. 10 μL of the stained cells were transferred into a
counting chamber slide and automatically counted.
Micro carriers
High concentration Synthemax®II-coated micro carriers (Corning) and CellBIND® were prepared
according to manufacturer’s instructions, as follows: A vial with dry sterile micro carriers was
transferred to a 100 mL bottle. The vial was washed twice with 10 mL growth medium. Finally 80 mL
of growth medium was added to the bottle with micro carriers and mixed by tilting the flask up and
down several times. The micro carrier stock solution was then stored at 4oC with a concentration of
100 mg/mL.
Cytodex 1 micro carriers were prepared as follows: The dry microcarriers are swollen and hydrated in
Ca2+ and Mg2+ free PBS (50100 ml/g Cytodex) for at least 3 h at room temperature. The supernatant
is decanted and the micro carriers are washed for a few minutes in fresh Ca2+ and Mg2+ free PBS (30
50 ml/g Cytodex). The PBS is discarded and replaced with fresh Ca2+ and Mg2+ free PBS (3050 ml/g
Cytodex and the micro carriers are sterilized by autoclaving (115 °C, 15 min, 15 psi). Prior to use, the
sterilized micro carriers are allowed to settle, the supernatant is decanted and the micro carriers are
briefly rinsed in warm culture medium (2050 ml/g Cytodex). When the micro carriers have settled,
the supernatant is discarded and the micro carriers are transferred to the culture vessel.
Micro carrier cell culture in spinner-flasks
Single cells from a 2D culture flask were transferred to a 125 mL spinner-flask (Corning) together with
5 mL micro carriers (100 mg/mL) and 45 mL growth medium. For attachment, the cells were
incubated with intermittent stirring (30 minutes rest, 3 minutes stirring at 50 rpm) at 37oC and 5 %
CO2 overnight. After cell attachment to micro carriers had occurred, growth medium was added to
double the volume, giving a volume of 100 mL. Continuous stirring was set to 50 rpm.
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For extended growth culturing, 5*106 culture new beads (183cm2) were cultured for 8 days and
samples were taken at day 1, 4, and 8 to follow the proliferation process. Both MTS and Qubit™
assays were performed for quantitative measurement and Hoechst fluorescent staining for
fluorescent microscopy. 1 mL of sample was taken for analysis. The protocol for the MTS assay was
adjusted by stirring the bead sample during incubation with the MTS reagent at 1000 rpm in an
orbital shaker. At day 8, three samples were taken to investigate reproducibility of the assays.
Bead-to-bead transfer was performed by adding fresh beads stained with Rhodamine 6G to cell
cultures at different time points. After 4 days of cell culture, beads were added, which we labeled
“early bead expansion culture”. The culture that was supplemented with new beads after 8 days was
named the “late bead expansion”. As a control, one culture was not supplemented with fresh beads,
the “continuous culture”. After adding the new beads, o/n intermittent agitation was switched on
(30 min rest, 3 spinning at 50 rpm). All conditions were performed in duplo. Samples were taken for
Qubit™ analysis, RNA analysis and microscopy at days 1, 2, 4, 6, 7, 8, 9 and 12. At day 12 the cells
were trypsinized and counted using Trypan blue exclusion method
Qubit™ dsDNA assay
Samples were prepared by transferring 1 mL of micro carrier-cell culture into a 1,5 mL Eppendorf
tube and spinning down for 1 minute at 1500 rpm. The supernatant was aspirated and the beads
with cells were washed with 1 mL PBS. Next, the beads were spun down for 1 minute at 1500 rpm.
The supernatant was aspirated and 100 µL RLT lysis buffer (Qiagen) was added. The samples were
incubated for 15 minutes at 55oC in an orbital shaker shaking at 1000 rpm. After incubation, the
samples were spun down for 1 minute at 1500 rpm. The DNA content was measured using the
Qubit™ dsDNA BR Assay Kit according to manufacturer’s instructions, as follows: Make the Qubit™
working solution by diluting the Qubit™ dsDNA BR reagent 1:200 in Qubit™ dsDNA BR buffer. Load
Qubit™ working solution into individual assay tubes so that the final volume in each tube after
adding sample is 200 µL. Add each of your samples to assay tubes containing the Qubit™ working
solution and mix by vortexing 23 seconds. The final volume in each tube should be 200 µL. Allow all
tubes to incubate at room temperature for 2 minutes. Insert a sample tube into the Qubit® 2.0
Fluorometer, close the lid, and press Read. Upon the completion of the measurement, the result will
be displayed on the screen.
MTS assay
To determine viable, metabolically active cells, MTS assay was performed. Samples were prepared by
transferring 1 mL of micro carrier-cell culture into a 1,5 mL Eppendorf tube and were spun down for
1 minute at 1500 rpm. The supernatant was aspirated and 100 µL of culture medium was added to
the samples. Next, 20 µL of CellTiter 96® AQueous One Solution Reagent was added into each
samples and incubated at 37oC. After two hrs incubation, the micro carriers were spun down to the
bottom and the supernatant was pipetted to a 96-well plate to be measured. The samples were
measured at absorbance 490 nm using a VICTOR3™ multilabel reader.
Hoechst staining
1,5 µl of Hoechst staining solution was added to 1 mL of micro carrier-cell culture sample. After 3
minutes of incubation, the sample was spun down for 1 minute at 1500 rpm. The supernatant was
aspirated and the beads with cells were washed with 1 mL PBS. Next, the beads were spun down for
1 minute at 1500 rpm. The supernatant was aspirated and the beads were transferred to a 24-wells
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plate by suspending the beads in 1 mL PBS and transferring the suspension to the plate. Next,
pictures were taken using fluorescence microscopy.
Rhodamin staining
500 μM Rhodamine was added to 5mL (100 mg/mL) beads and incubated for 25 minutes. The beads
were then washed with PBS three times and the labelling was confirmed by fluorescence microscopy.
Labeled beads were added to the existing culture and were incubated with intermittent stirring (30
minutes rest, 3 minutes stirring) at 37oC and 5 % CO2 overnight.
EDTA detachment
A titer of EDTA concentrations was made ranging from 50mM to 1µM dissolved in MiliQ (pH7). Cells
were seeded on a 6-wells plate coated with Synthemax and the next day the EDTA was added and
incubated for 1.5 hrs. After incubation, the supernatant was transferred to a newly coated 6-wells
plate. To both the original and transferred plate, 2 ml of growth medium was added. After 3 days the
cells were assessed for cell growth using microscopy.
RNA isolation and Q-PCR
2 mL bead-cell suspension samples were taken for Q-PCR analysis. The samples were spun down for
1 minute at 1500 rpm. The supernatant was aspirated and the beads with cells were washed with 1
mL PBS. Next, the beads were spun down for 1 minute at 1500 rpm. The supernatant was aspirated
and 350 µl of RLT lysis buffer was added to the cells and was frozen at -20oC. For RNA isolation 350 µl
of 70% Ethanol was added to the sample. Next, the RNeasy Mini Kit (Qiagen) was used to isolate the
RNA according to manufactures protocol.
After RNA isolation Q-PCR was performed by pipetting 4 µl SYBR Green Supermix-UNG, 1 µl Primer
(10 µM) forward + reverse, 3 µl RNase-free MQ for primer master mix and 4 µl SYBR Green Supermix-
UNG, 1 µl cDNA template and 3 µl RNase free MiliQ for cDNA master mix per reaction.
Q-PCR protocol: 5:00 min 45oC, 2:00 min 95oC, 0:15 min 95oC 40X, 1:00 min 95oC, 1:00 min 65oC,
0:10 min 65oC 60X, 0:30 min 20oC.
Table 1. Primer sequences used for Q-PCR.
Gene
Fwd Sequence
Rev Sequence
GAPDH
TCC-CAA-CGT-GTC-TGT-TGT-GGA-TCT
TGT-TGA-AGT-CGC-AGG-AGA-CAA-CCT
Beta Actin
GGC-ACC-CAG-CAC-AAT-GAA-GAT-CAA
ATC-GTA-CTC-CTG-CTT-GCT-GAT-CCA
Cyclophilin
CCC-ACC-GTG-TTC-TTC-GAC-AT
CCT-TGT-CTG-CAA-ACA-GCT-CAA-A
MyoD
TAG-GAG-AGG-CGA-AGG-AAC-TGT-TGT
TCT-GGC-CCA-CGG-AGT-AAC-ATC-AAA
Myogenin
AGC-CTC-CAA-ATC-CAC-TCC-CTG-AAA
AGC-CAC-TGG-CAT-AGG-AAG-AGA-TGA
Statistics
All analyses were made with at least three replicates of each sample. SPSS was used to evaluate
statistical evidence. Experiments were evaluated by one way ANOVA. Significant difference was
determined at p<0.05.
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Results
Cell inoculation density on micro carrier experiment
For cells other than satellite cells, seeding density determines growth rate (Ng et al. 1996), but the
optimum has not been described for satellite cells yet. We seeded different amounts of cells (1*106,
5*106, 1*107) on a fixed amount of beads (183 cm2) to determine the optimal seeding density in
terms of growth and homogeneity of bead colonization.
For measuring cell number, two different assays were used. The MTS assay based on metabolic
activity of cells (figure 4-A) and the Qubit™ dsDNA assay (figure 4-B) for measuring the amount of
DNA were performed.
The MTS assay failed to show differences between cell growth as a function of inoculation densities
(NS). In contrast, when analyzing the Qubit™ assay, differences were observed between the cell
inoculation densities (p<0.05). The differences between the values were unfortunately not
proportional with the increase of cells added to the culture (5*106 was not 5 times larger than 1*106,
1*107 was not twice as large as 5*106).
To address this possibility, the attachment efficiency was investigated in a separate experiment
where 3*106 cells were inoculated onto the beads overnight. The next day, the beads, medium and
the bottom of the spinner flask were lysed and measured with the Qubit™ assay to investigate the
attachment efficiency of the cells (figure 4-C). From the total amount of DNA measured, 67%
originated from the beads, 33% was not bound to the beads. This “loss” of DNA could be an
explanation for the inconsistency we observed between the 1, 5 and 1*107 inoculation cell density.
When the cell density exceeds the available space on the micro carriers, the amount of cells bound to
the beads will likely decrease resulting in disproportionally low DNA measurements on sampled
beads.
For microscopy, cells were stained with Hoechst fluorescent dye adhering to DNA (figure 4-D). The
pictures were taken on days 1, 2 and 4. The high-density inoculation (1*107) resulted in the
formation of aggregates of beads connected by a cell cluster. On the other hand the 1*106 condition
showed little growth of the cells with exception of a few beads that were highly populated at day 4
(figure 4-D). Based on this finding we decided to continue further studies with cell densities between
1*106 cells and 5*106.
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Figure 4. Cell numbers at day 4 with different inoculation-densities
(n=4 for each) as measured by MTS (A) and Qubit (B), One way
ANOVA analysis showed no significant difference between the three
inoculation densities at the MTS assay but significant difference can
be seen in the Qubit assay. (C): Qubit measurements of different
culture components (beads, medium, and bottom of spinner flask)
after 24 hr attachment. (D) Photomicrographs of Hoechst stained
micro carriers with different cells densities. Photos were taken at day
1, 2 and 4.
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Cell inoculation density experiment with extended growth
Previous data has shown that an inoculation density between 1*106 and 5*106 is preferred over
higher cell numbers. To further investigate what cell amounts are optimal for long-term growth we
repeated the previous experiment using 1*106, 3*106, and 5*106 cells to inoculate the micro carriers.
According to the MTS assay, at day 8, a significant difference (p<0.05) was detected between the
different inoculation cultures. Unfortunately no increase in absorbance was measured during the 8
days of culturing, which would suggest that no proliferation had occurred. The microscope photos
(figure 5 F) showed a clear increase in cell number over time.
The Qubit assay shown in figure 5 B shows a significant increase of DNA between the inoculation
cultures (p<0.05) and also an increase in DNA concentration over the culture period. These findings
correlate with the pictures that were taken (figure 5 F). In these pictures there is an expansion of
cells visible on the micro carriers, which indicates proliferation of the cells. There is an increase in
empty beads visible in the picture of the 5*106 culture on day 8, which is likely caused by the
addition of new beads on day 4. No cell transfer to the new beads was apparent, which corresponds
with the Qubit data where no notable increase of DNA was observed.
To inspect the distribution of cells onto the micro carriers, we used the pictures that were taken from
the Hoechst staining and counted the amount of cells per bead on 80 randomly selected beads
(figure 5-C thru E). The 1*106 culture (figure 5-C) had a large amount of empty beads during the
culturing period. Interestingly, the amount of beads that housed 15 or more cells increased about 10-
fold until day 8. Furthermore a bimodal distribution is observed with an abundance of beads with 0-5
cells per bead and the highly populated beads at the top end of the distribution.
The cells-per-bead distribution in the 3*106 culture seemed normal with a maximum at 5 cells per
bead and the isolated peak at 15 or more cells per bead, with a shift to the right at day 8. A similar
pattern in the 5*106 was visible only with more beads containing more than 15 cells. The distribution
of this culture was only counted till day 4 because after this point new beads were added.
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Figure 5. (A) Measurements of micro carrier cell culture, with different inoculation, during 8 days
of culturing measured with MTS assay (A) and Qubit assay (B). When analyzed using a one way
ANOVA all values at day 8 are significant different. Significance between day 1, 4 and 8 could
not be determined since no replicates were taken (C, D, E) Distribution of cell onto micro
carriers. Note that day 8 data are missing from panel E, because new beads were added in this
experiment. (F) Hoechst staining of micro carriers with different cells densities. Photos were
taken at day 1, 4 and 8.
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Detachment of cells from Synthemax coating (2D) using
Ethylenediaminetetra acetic acid (EDTA)
When cells are grown on a micro carrier system they need to be separated from the beads before
using them to make muscle fibers. The beads are made of polystyrene and cannot be incorporated
into the final meat product. The commonly used enzyme trypsin for this is expensive and not very
effective. Here we suggest an enzyme-free method for detaching cells from micro carriers using only
EDTA.
Results were qualitatively assessed from photomicrographs of cultures (Figure 6) before and after
EDTA incubation at various concentrations. In figure 6 the pictures are shown of the cells during the
experiment. In picture A, the plate is shown after o/n seeding of the cells and the left row of pictures
show the 25mM condition. This shows a normal cell attachment and cell morphology. After 1 hr of
incubation with the EDTA the 50mM and 25mM show signs of detachment, which is visible by the
light circles around the cells. This is also visible at the 1mM condition but to a lesser extent. Row B
shows the cells after 1,5 hr of incubation with a fair amount of cells being detached from the plate
surface. At 50mM EDTA most cells were detached and the least at the 1mM condition. Next the
supernatant was transferred to a new 6-wells plate (the transfer plate) and 2 mL fresh growth
medium was added to both conditions.
After 3 days both plates were assessed for cell growth. Photos were taken of the original plate and
the transfer plate (figure 6 D and E respectively). At 50mM, cells in the original plate (row D) were
growth impaired compared to the 25mM and 1mM condition EDTA treatment, with the latter
showing a fully grown plate. After 1mM EDTA however, the cells showed a strange morphology with
cells in a largely stretched phenotype (white arrows). Unfortunately the transfer plates showed
hardly any cells. By destabilizing adhesion proteins we hoped to detach the cells from their surface
without using trypsin. All other conditions gave negative result (data not shown).
17
Figure 6. (A) Cells seeded on 6-wells plate after o/n attachment. (B) One hr of incubation with EDTA,
50mM, 25mM and 1mM, is shown. Moderate detachment is visible. (C) Incubation time of 1,5 hrs
shows highest detachment at the 50mM condition and least At 1mM condition. (D) After 3 days
recovery the 50mM condition shows impaired growth compared to the 25mM condition. The plate of
the 1mM condition is grown to confluence, however some cells show divergent morphology with long
stretches (white arrows). (E) The transfer plates show hardly any cells indicating a failed transfer of
the cells to the new plate.
18
Bead-to-bead transfer experiment
As expected, DNA content as measure of number of cells increased over time. Interestingly, sudden
and robust increases were observed after adding new beads at days 4 and 8, which clearly were not
detected in the continuous culture (Figure 7-A). By staining the newly added beads with Rhodamine
6G, we directly showed that the new beads were covered by cells (figure 7-E). To rule out an effect
of the beads on the Qubit assay empty beads and empty beads with an added DNA standard that
was included in the kit for calibration were measured. No effects on the outcome of the DNA
measurement was observed (data not shown).
Cells were detached and counted at day 12 (figure 7-B). More cells per milliliter were counted in the
late bead expansion culture compared to the early bead expansion and continuous culture. After
trypsinization, the beads were stained using Hoechst for confirmation of cell detachment (figure 7-E).
The results show that a substantial portion of the cells still were attached to the beads, which
obviously impacted the outcome of the analysis and is undesirable if cells are to be harvested for
muscle fiber production.
During the culturing period gene expression of MyoD and Myogenin were studied. These genes are
associated with proliferation and differentiation respectively. Gene expression in both genes
dropped after day 4 in all conditions. While DNA concentrations rose in the early and late bead
expansion conditions, RNA expression for these genes declined.
19
Figure 7. (A) Growth curve of 3 cell cultures during 12 days period (n=2). Beads were added to the early bead
expansion at day 4 (red line). Increase of DNA content was measured at day 6. Late bead expansion (green line) was
supplemented with beads at day 8. Similar increase of DNA was measured at day 9. No increase of DNA was
observed in the continuous culture (blue line). (B) Cell amount after trypsinization measured using Trypan blue
exclusion method. (C, D) Expression of MyoD and Myogenine during 12 days of culturing. (E) Photographs of the
cell cultures stained with Hoechst at day 1, 8 and 12. Rhodamine 6G stained beads are visible at day 8 in the early
bead expansion condition. The same was observed at day 12 the late bead expansion group. The presence of cells
on stained beads is a clear indication of bead-to-bead transfer. Bottom 3 photos were taken after trypsinization.
20
Micro carrier variation experiment
Cytodex, Cellbind and Synthemax beads were compared for growth efficiency. The Cytodex beads
showed the highest DNA concentrations compared to the Synthemax and Cellbind. Cellbind and
Synthemax showed similar DNA concentrations. DNA concentrations in the Cytodex conditions had a
difference of 0,188 µg/mL at day 1.
In figure 8-B an attachment experiment was performed. When 1 million cells are used 95% attach to
the beads with only a 5% loss. With 3 million cells are large amount of cells does not attach the beads
and end up in the medium or at the bottom of the spinner flask. For the following experiments 1
million cells was used instead of the 3 million cells that were used for previous experiments.
Next, the bead-to-bead transfer experiment was performed. Fresh beads were added on day 3 and 6
to the early and late bead expansion respectfully. In figure 8-C the growth curve is shown. At day 3 a
rise of DNA concentration is observed in de early bead expansion culture. In the late bead expansion
culture a similar rise is observed at day 6. However, a decline of DNA concentration is visible in both
continuous and the late bead expansion culture at day 3. When beads grow confluent the satellite
cells form large aggregates, which detach from the beads.
After 9 days the satellite cells were detached from the micro carriers and cultured onto a 6-wells
plate to investigate if the cells were still able to differentiate after growing on beads (figure 8-E.) In
the continuous culture no myotubes were formed after 6 days of differentiation. In the early and late
bead expansion culture some myotubes were formed (white arrows) while this was less than
expected.
21
Figure 8. (A) Growth curve of cells on Cytodex, Synthemax or Cellbind micro carriers. 1 or 3 million cells were seeded
on 183cm2 of bead and cultures for 8 days. When using Cytodex, higher DNA concentrations were measured than
when using Synthemax or Cellbind. (B) Attachment efficiency of Cytodex micro carriers. When using 1 million cells
95% of the cells attach to the bead where only 52% of the cells bind to the beads when 1 million cells are used. (C)
Growth curve of bead-to-bead experiment. New beads were added on day 3 (early bead expansion) and day 6 (late
bead expansion). No beads were added to the continuous culture. A robust rise in DNA concentration is observed
when beads are added to the culture. (D) Supporting fluorescent photos of the bead-to-bead experiment. On day 9
of the continuous culture an increase of empty bead is visible. (E) At day 9 of the bead-to-bead experiment the cells
were detached and transferred to a 6-wells plate to investigate the differentiation capabilities of the satellite cells
after 9 days of growth on beads. The continuous culture shows no myotube formation while the early and late bead
expansion cultures show some (white arrows).
22
Discussion
In this study we have shown that bovine satellite cells can proliferate and expand on non-animal
derived surfaces. This is a crucial step towards cell expansions without the need of animal based
resources, with the exception other than the satellite cells themselves. To make in vitro meat a
realistic solution to the worlds demand for meat, more hurdles must be overcome like scaling,
resource renewability, and physical sensations just to name a few. Even though these may be
challenging, this also gives rise to numerous opportunities for innovation and progress, not only for
this field. Novel hydrogel production (Rahimi 2012) and cell culture optimizations (this report) are
discoveries that can be used for meat production but can also be applied to other fields like organ
tissue engineering.
In the cell inoculation experiment, the need to find a balance between cells and bead surface was an
important step in scaling this technique. We found that too little cells, 1*106 / 183cm2 of Synthemax
beads, was insufficient to colonize every bead. The low cells amount also seemed to have an effect of
cell growth behavior, were the cells localized to some beads were others were deserted or cells
didn’t proliferate. The halt in the proliferation may be caused due to the lack of cell-cell contact
(Mather, Roberts 1998). When a “high” amount of cells were used (10*106) aggregates were formed.
Ferrari et al. determined the high cell density to be the cause. This is undesirable because it likely
inhibits the growth of cells by steric hindrance or reduction in oxygen and nutrient delivery (Sen et
al.). The 5*106 cell density colonized al the beads. However, it is advantageous to have an as low as
possible starting amount to minimize the amount of cells that is needed to start a bead culture.
Furthermore, DNA concentrations were not proportional with the amount of cells that were used.
This inconsistency could be caused by non-adherence of the cells to the beads, but adherence for
instance to the wall or bottom of the flask.
Next, the experiment was repeated with lower inoculation densities. Here was shown that 3*106
cells also colonized all the beads and gave a similar distribution pattern. When growth was tracked,
using the Qubit analyses, the 5*106 inoculation density didn’t show a large difference in DNA
concentration compared to the 3*106 condition. The value of the 5*106 culture at day 4 is probably
caused by a measuring error that could occur when micro carriers are accidentally pipetted into the
96-wells plate. Because of the consistently poor performance of the MTS assay we decided that the
assay is not a suitable method for measuring cell amount and expansion of satellite cells on micro
carriers. The reason why this assay performs poorly is not clear. To minimize inoculation density, the
3*106 condition was selected for following experiments.
After expanding the culture on the micro carriers, the cells need to be detached from the beads for
further processing. For small scale culturing trypsin is used. For large scale culturing it would be
undesirable to use large amounts of trypsin. By using EDTA we tried to destabilize the cell anchor
proteins to detach them from the beads. This was suggested on the website: protocol-online.org or
using citric acid (The Carver College of Medicine 2007). Unfortunately it was unsuccessful. The
satellite cells did detach but didn’t survive the transfer to a new plate. The reason why the cells
didn’t survive is still unclear.
23
To realize mass production of satellite cells, a labor convenient system is preferable. A fed-batch
system would be suitable for this goal. In this system new beads can be added to a near confluent
culture. Agitated stirring brings fresh beads in close contact with beads containing cells, thus
enabling cell transfer (Wang 1999). This way, when using a bioreactor, only fresh medium and beads
can be added to a closed system thus minimizing the risk of infection, aggregate formation (Ferrari
2012) and handling.
In the first bead-to-bead experiment, new beads were added at day 4 to the early bead expansion
culture, at day 8 in the late bead expansion, and no fresh beads were added to the continuous
culture. When the beads were added o/n intermittent agitation was switched on. When the DNA
concentration was measured the next day, a sharp increase was observed in the culture where the
beads were added. The transfer of cells to fresh beads was confirmed my microscopy. However, the
increase of DNA was so large that it was questionable if this was a direct cause of cell proliferation
onto the added beads, or could be caused by cells previously attached to the bottom of the spinner
flask, which attached to the new beads. Nonetheless, this increase was robust with a similar increase
found in the late bead expansion. No sharp increase of DNA content was found in the continuous
culture.
At day 12 the cells were detached using trypsin for counting. This showed a higher cell count in the
late bead expansion compared to the late and continuous culture. However, when the beads were
stained after detachment, it showed that a substantial portion of the cells still are attached to the
beads which impacts the outcome of the analysis and is undesirable if cells are to be harvested for
muscle fiber production. Communication with manufacturer revealed that these micro carriers
attract serum, which inhibits dissociation when trypsin is used. Hopefully this will be resolved when
cells are grown under serum-free conditions which are currently being investigated.
During the culturing period samples were taken for RNA analysis. MyoD and Myogenin were studied.
These genes are associated with proliferation and differentiation respectively. Over time the gene
expression declined for both genes. We expected MyoD expression to decline and Myogenin
expression to increase. A reason for this to happen is dedifferentiation of the cell population (Slack
2006). This may cause the satellite cells to lose expression of specific genes that would be used as
markers for proliferation and differentiation (Yin 2013). Because of this, no time point during
culturing could be determined for adding new beads to the cell culture to keep cells in a proliferate
state.
When Cytodex1, Synthemax, and Cellbind were compared during a proliferation assay, Cytodex1 was
found to be the most suitable for satellite cells expansion. The attachment assay that was performed
in parallel also revealed that only 1 million cells was sufficient to colonize the Cytodex1 beads. The
cause of this probably lies in the attachment capabilities of the cells to the beads. Adding 3 million
cells resulted in only a fraction more attached cells, and a higher number of unattached cells and
would therefore be wasted. Next the bead-to bead experiment was repeated using the Cytodex1
beads. Again a robust rise in DNA concentration was observed when new beads were added. What
this experiment also showed was a decline in DNA when no beads were added. On day 9 of the
continuous culture an increase of empty bead is visible. This is probably the cause for the decrease of
DNA concentration which may be caused by detachment of confluent cells, and could be prevented
by adding beads at the right moment (Ferrari 2012).
24
After 9 days the satellite cells were detached from the micro carriers, using trypsin, and were seeded
on to a 6-wells plate. Here we cultured the cells till confluence and initiated differentiation. No
myotubes were formed in the continuous culture condition. This condition received no additional
beads which may have led the cells to differentiate during culturing. Once differentiation has
occurred, the cells do not divide and the nuclei never again replicate their DNA (Molecular Biology of
the Cell, 5th Edition). The early and late bead expansion conditions showed little myotube formation.
This could be caused by a high population doubling during culturing. To investigate how many
population doublings the cells undergo during culturing, telomere length could be studied. This
would give insight into aging of the cells during growth on beads since there is no clear passage
number.
Conclusion
To summarize the findings in this report:
1. When Sythemax micro carriers are used to grow satellite cells, an inoculation density of
3*10^6 per 183cm2 is optimal to prevent aggregates and colonize all the beads.
2. For tracking cell growth the MTS assay is not suited for this application because of the
inconsistent results. The Qubit™ assay however, concords with the visual counting of cells.
This makes it suitable for measuring cell growth in this application.
3. Bead to bead transfer was achieved by adding new beads overnight with an intermitted
stirring program.
4. Using EDTA to detach cells from micro carriers is was not successful.
5. When Cytodex beads are used, higher DNA concentrations are achieved compared to using
Synthemax or Cellbind micro carriers.
6. After 9 days of culturing on micro carriers, satellite cells are still capable to differentiate into
myotubes after detachment.
25
Future Prospect
There is still a long way before in vitro meat becomes a credible alternative to livestock meat, never
the less concerns over environmental, sustainability and animal welfare are growing each year (FAO
2006). Possibilities in personal health are also a benefit to this development, for example: the
nutritional value of meat might be altered to make it a healthier or specialized diet product, for
instance by increasing the content of poly-unsaturated fatty acids through changes in culture
conditions (Post 2012). More research is needed to overcome the bottlenecks that are limiting the
adaptation of in vitro meat into society.
One of the bottlenecks to this technique is the use of animal derived serum. With recent
development in microalgae genetic engineering, this group of organisms could be a suitable and
sustainable candidate to produce animal based medium (Pulz, Gross 2004). Also when culturing
satellite cells for a prolonged period, problems with cell aging could occur. This may cause the cells to
lose their myotube forming ability. A solution to this problem is still socially undesired. When satellite
cells are immortalized, they could be an infinite source of meat. However, the effect of
immortalization to differentiation is still unknown and when it comes to genetically modified food,
the general population is still very conflicted.
Another limitation for mass production in vitro meat is the formation of tissue from the cultured
cells. For making an 85 gram hamburger 10.000 muscle strips (BAM) need to be produced. This
method is laborious and likely not suited for large scale production. Larger pieces of tissue would be
advantageous but these would be limited by the dependence on an adequate nutrient and oxygen
supply through diffusion (100µm) (MacDougall 1967). No attempts have been made yet to create
large BAMs with a built-in blood vessel or channel system conducting a continuous flow of
oxygenized, nutrient-rich medium. A novel approach is suggested using a combination of state of the
art 3D printers in combination with hydrogels.
In 2012 Jordan S. Miller published a method to produce a vascular network into a hydrogel. By 3D
printing a sacrificial filament network made of carbohydrate glass, a mold could be produced. When
the lattice mold was encapsulated with a hydrogel-cell suspension the carbohydrate scaffold was
dissolved using water which left smooth elliptical intervessel junctions that supported fluidic
connection between adjoining vascular channels. These channels were seeded with endothelial cells
(HUVECs) which resulted in the lining of the entire network. Next this network was perfused with
human blood and was cultured for 9 days. Optical sections showed a higher concentration of cells
compared to gels without channels (Miller 2012).
This technique could also be implemented for the production of in vitro meat, with some
enhancements. For example an edible hydrogel should be used if the constructs are to be used for
consumption, which should also be capable to undergo electrical stimulation for better distribution
of the extra cellular matrix (ECM) (Rahimi 2012) and in vitro exercise to boost protein content of the
cells (Nataša Nikolić 2012).
Besides satellite cells, adipose cells should be co-cultured to improve the experience to be on par
with conventional meat. When using this system this becomes possible by mixing these cells with the
satellite cells into the hydrogel. What the effect of the adipose cells is on the satellite cells is still a
26
topic of research. When using a setup depicted in figure 8 the limitation of oxygen and nutrient
diffusion could be overcome. Moreover, this system allows for a pulsating flow through the tissue,
which mimics the normal physiological condition in the body. This has been shown to positively
affect proliferation during culturing (Mun 2013).
Figure 9. Schematic drawing of in vitro beef culture system using a sacrificial blood vessel mold made
from carbohydrate. Using this system allows for “large” 3D tissue production which is desirable when
upscaling production.
27
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Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T-flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex-1®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50×10(6) cell g(-1) Cytodex-1®). Then, the MSC aggregates were put into contact with additional available surfaces (T-flask, fresh and used Cytodex-1®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T-flasks or fresh Cytodex-1® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex-1® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex-1® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number.
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Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.