U.S. patent application number 17/831351 was filed with the patent office on 2022-09-22 for methods for improving cell growth with species-specific or genus-specific proteins and the applications thereof.
This patent application is currently assigned to Avant Meats Company Limited. The applicant listed for this patent is Avant Meats Company Limited. Invention is credited to Kai Yie Carrie CHAN, Po San Mario CHIN, Chun Hei POON, Lok Hin WON.
Application Number | 20220298480 17/831351 |
Document ID | / |
Family ID | 1000006434050 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220298480 |
Kind Code |
A1 |
CHIN; Po San Mario ; et
al. |
September 22, 2022 |
METHODS FOR IMPROVING CELL GROWTH WITH SPECIES-SPECIFIC OR
GENUS-SPECIFIC PROTEINS AND THE APPLICATIONS THEREOF
Abstract
A method for meat production by in vitro cell culture includes
isolating tissue from an animal or plant source and making a cell
suspension of cells, and growing the cells into a solid or
semi-solid structure that mimics an animal organ by growing the
cells on a food-grade scaffold in a culture medium. Culture medium
comprising growth factor of (i) genetically same or similar species
to the cells and/or (ii) genetically same genus to the cells is
used. Expression of one or more proteins in the growing cells may
be increased by altering a level of one or more micro RNAs that
regulate expression of the protein. Additionally, the growing cells
may be co-cultured with bioengineered cells that secrete growth
factors and cytokines that support the growth of the cells in situ.
The co-culturing technique reduces or eliminates the need for
animal-derived fetal bovine serum in the culture medium.
Inventors: |
CHIN; Po San Mario; (Hong
Kong, CN) ; CHAN; Kai Yie Carrie; (Hong Kong, CN)
; POON; Chun Hei; (Hong Kong, CN) ; WON; Lok
Hin; (Hong Kong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Avant Meats Company Limited |
Hong Kong |
|
CN |
|
|
Assignee: |
Avant Meats Company Limited
Hong Kong
CN
|
Family ID: |
1000006434050 |
Appl. No.: |
17/831351 |
Filed: |
June 2, 2022 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/IB2020/061200 |
Nov 27, 2020 |
|
|
|
17831351 |
|
|
|
|
62942568 |
Dec 2, 2019 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 13/00 20160801;
C12N 5/0679 20130101; C12N 2501/105 20130101; C12N 2501/115
20130101; C12N 2501/2311 20130101; C12N 2501/11 20130101; C12N
2501/33 20130101; C12N 2533/74 20130101; C12N 5/0696 20130101; C12N
5/0652 20130101; A23L 17/00 20160801; C12N 2501/998 20130101; C12N
2501/2306 20130101; A23V 2002/00 20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; C12N 5/071 20060101 C12N005/071; C12N 5/074 20060101
C12N005/074; A23L 13/00 20060101 A23L013/00; A23L 17/00 20060101
A23L017/00 |
Claims
1. A method for meat production by in vitro cell culture,
comprising: isolating a tissue from an animal or plant source and
making a cell suspension of cells therefrom; introducing culture
medium comprising one or more growth factors of (i) genetically
same or similar species to the suspension of cells and/or (ii)
genetically same genus to the suspension of cells; and growing the
suspension of cells on a food-grade scaffold in a culture medium,
whereby the suspension of cells grows into a solid or semi-solid
structure that mimics an animal organ.
2. The method of claim 1, wherein isolating the tissue comprises
isolating an organ tissue from a fish.
3. The method of claim 1, further comprising the step of increasing
expression of a protein in the suspension of cells by altering a
level of one or more micro RNAs that regulate the expression of the
protein.
4. The method of claim 2, wherein the organ tissue is derived from
fish swim bladder of a fish from the Osteichthyes class.
5. The method of claim 3, wherein the protein is collagen.
6. The method of claim 3, wherein the micro RNAs are one or both of
micro RNA21 (miR-21) and micro RNA 29a (miR-29a).
7. The method of claim 1, wherein the growth factor is selected
from the group consisting of insulin growth factor 1 (IGF-1),
insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R),
interleukin 11 (IL-11), fibroblast growth factor (FGF), epidermal
growth factor (EGF), and transferrin.
8. The method of claim 1, wherein the growth factor to be
introduced to the cell culture medium is obtained from a
recombinant cell comprising a gene of the growth factor, wherein
the gene of the growth factor is of (i) genetically same or similar
species to the suspension of cells and/or (ii) genetically same
genus to the suspension of cells.
9. The method of claim 8, wherein the recombinant cell is a cell
from a prokaryotic organism or an eukaryotic organism.
10. The method of claim 8, wherein the recombinant cell is a yeast
cell.
11. The method of claim 2, wherein the growth factor to be
introduced to the cell culture medium is obtained from a
recombinant cell comprising a gene of the growth factor, wherein
the gene of the growth factor is of (i) genetically same or similar
species to the fish from which the organ tissue is isolated and/or
(ii) genetically same genus to fish from which the organ tissue is
isolated.
12. The method of claim 11, wherein the recombinant cell is a cell
from a prokaryotic organism or an eukaryotic organism.
13. The method of claim 11, wherein the recombinant cell is a yeast
cell.
14. A method for meat production by in vitro cell culture,
comprising: isolating a tissue from an animal or plant source and
making a cell suspension of cells therefrom; growing the suspension
of cells on a food-grade scaffold in a culture medium, whereby the
suspension of cells grows into a solid or semi-solid structure that
mimics an animal organ; and co-culturing the suspension of cells
with a plurality of bioengineered cells that secrete nutrients,
growth factors, and/or cytokines that support the growth of the
suspension of cells, wherein the bioengineered cells is (i)
genetically same or similar species to the cells and/or (ii)
genetically same genus to the cells.
15. The method of claim 14, wherein isolating the tissue comprises
isolating an organ tissue from a fish.
16. The method of claim 15, wherein the organ tissue is derived
from fish swim bladder of a fish from the Osteichthyes class.
17. The method of claim 14, wherein the growth factor is selected
from the group consisting of insulin growth factor 1 (IGF-1),
insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R),
interleukin 11 (IL-11), fibroblast growth factor (FGF), epidermal
growth factor (EGF), and transferrin.
18. The method of claim 14, wherein the suspension of cells is fish
swim bladder cells and the bioengineered cells are fish cells.
Description
TECHNICAL FIELD
[0001] Embodiments discussed herein generally relate to improved
methods for meat production using in vitro cell culture.
Embodiments discussed herein also generally relate to the improved
methods for cell growth using growth factors.
BACKGROUND
[0002] Animal meat is high in protein, and supplies all the amino
acids needed to build the protein used to support body functions.
Meat for consumption is traditionally obtained from animals or fish
that are reared on farms. However, agriculture and aquaculture for
producing animal meat require a large amount of energy and
resources, and have a high carbon footprint. Meat produced by
agriculture or aquaculture may pose a public health risk as the
production processes may expose the meat to diseases, pollutants,
and toxins. A number of concerns such as a growing population,
increasing demand for meat, environmental concerns, limited land
and water resources, biodiversity loss, and the negative perception
associated with animal slaughter have led scientists to develop
techniques to produce meat by alternative processes.
[0003] In vitro meat production is the process by which muscle
tissue or organ tissue from animals are grown in laboratories using
cell culture techniques to manufacture meat and meat products. As
used herein, in vitro meat and meat products includes animal
protein products as well as non-meat products including soluble
forms and solid forms. While still in an early stage of
development, in vitro meat and meat products may offer a number of
advantages over traditional meat products such as health and
environmental advantages, and benefits to animal welfare. It is a
next-generation and emerging technology that operates as part of a
wider field of cellular agriculture, or the production of
agricultural products from cell cultures.
[0004] Cells for the production of in vitro meat may be cells
(e.g., muscle cells, somatic cells, stem cells, etc.) taken from
animal biopsies, which may then be grown separately from the animal
in culture media in a bioreactor or other type of sterile
environment. The cells may grow into a semi-solid or solid form
mimicking an animal organ by attaching to an edible
three-dimensional scaffold that is placed in the bioreactor. The
starter cells may be primary cells directly obtained from the
animal's tissues, or continuous cell lines. If grown under the
right conditions in appropriate culture media, primary cells will
grow and proliferate, but only a finite number of times that is
related to the telomere length at the end of the cell's DNA.
Continuous cell lines, on the other hand, can be cultured in vitro
over an extended period. Cell biology research has established
procedures on how to convert primary cells into immortal continuous
cell lines. Primary cells may be transformed into continuous cell
lines using viral oncogenes, chemical treatments, or overexpression
of telomerase reverse transcriptase to prevent the telomeres from
shortening.
[0005] The culture media may contain components necessary for cell
proliferation such as amino acids, salts, vitamins, growth factors,
and buffering systems to control pH. Current methods add fetal
bovine serum (FBS) to the media prior to use as it provides vital
macromolecules, growth factors, and immune molecules. However, FBS
is derived from unborn calves and, therefore, is incompatible with
the objective of being free from animal products. Growing the cells
in an animal component-free medium is an important factor
considered by scientists involved in in vitro meat production
research. Some growth factors may be derived from human
sources.
[0006] Generally, over 95% of the culture-medium cost is attributed
to the protein components. Recombinant human growth factors (e.g.
insulin, IGF-1), human serum albumin (HSA), or fetal bovine serum
(FBS) are often supplemented in excess amounts to basal media.
While human protein factors and FBS effectively promote the growth
and differentiation of human cells, they are less bioactive on
cells from distant species (e.g. fish, bird). This causes a long
culturing period and low cell quality. To compensate for the low
bioactivity of non-human cells, excessively high levels of human
protein factors or FBS are added to the growth medium, which leads
to high costs.
[0007] Current in vitro meat production covers most commodity meat
types, such as cell-based beef, pork and poultry meats. However,
these types of meats have a complex tissue organization involving
multiple cell types that are difficult and costly to produce using
current biomedical technology techniques. There is also a lack of
non-GM methods to increase the protein level and biomass yield in
meat produced by cell culture techniques. Furthermore, as explained
above, current cell culture technologies may rely on animal
components (e.g., FBS) as a nutrient source, as well as expensive
non-food grade growth factors.
SUMMARY
[0008] The embodiments of the present disclosure apply methods for
in vitro meat production for human consumption that provides a
solution to the above challenges.
[0009] It is an objective of the present invention to provide an
alternative method to cultivate cells using species-specific or
genus-specific growth factors. This approach not only decreases
medium-cost by lowering growth factor usage, but it also shortens
the culturing time and improves cell quality by enhancing cellular
responses. Using species-specific or genus-specific growth factors
may help to enhance cellular response (for example, growth,
differentiation) and break the maximum cellular response
encountered when using non-species-specific or non-genus-specific
growth factors.
[0010] It is also an objective of the present invention to provide
a method to evaluate the efficacies of growth factors of different
species origins in stimulating cell growth.
[0011] According to one embodiment of the present disclosure, a
method for meat production by in vitro cell culture includes
isolating tissue from an animal or plant source and making a cell
suspension of cells. The method further includes introducing
culture medium comprising growth factors of (i) genetically same or
similar species to the cells and/or (ii) genetically same genus to
the cells. Additionally, the method further includes growing the
cells on a food-grade scaffold in a culture medium, the cells
growing into a solid or semi-solid structure that mimics an animal
organ.
[0012] According to another embodiment of the present disclosure, a
method for meat production by in vitro cell culture includes
isolating tissue from a plant or animal source and making a cell
suspension of cells, and growing the cells on a food-grade scaffold
in a culture medium such that the cells grow into a solid or
semi-solid structure that mimics an animal organ. The method
further includes co-culturing the cells with bioengineered cells
that secrete nutrients, growth factors, and cytokines that support
the growth of the cells, wherein the bioengineered cells are (i)
genetically same or similar species to the cells and/or (ii)
genetically same genus to the cells.
[0013] In some embodiments, the species is genetically similar to
the cells when they are more than 90% match in DNA sequence.
[0014] Embodiments disclosed herein apply methods for in vitro meat
production for human consumption that provides a solution to the
above challenges.
[0015] In a further embodiment, the growth factor that supports
cell growth can be produced from microorganisms containing and
expressing a transgene of the growth factor. The microorganisms may
be bacteria, yeasts or fungi. Given the fact that purification of
the expressed growth factor from the microorganisms involves a
complicated and expensive process, embodiments of the invention
develop a process to use the growth factor directly from the yeast
culture to culture fish cells without an elaborated purification
procedure. Aspects of the invention may genetically engineer a
yeast clone that contains a fish IGF-1 gene. In one aspect, the
recombinant yeasts may be grown in a yeast culture medium to allow
protein expression of the IGF-1 gene. In one aspect, the IGF-1 may
be secreted into the yeast culture medium. In another example, the
IGF-1-containing yeast culture medium may be centrifuged at about
1500 g for 5 minutes to separate the yeast from the medium. The
supernatant may be collected and centrifuged again at about 7200 g
for about 15 minutes at about 4.degree. C. The clarified
supernatant may then be filtered with a 2 .mu.m or smaller pore
size filter. The filtered IGF-1-containing yeast culture medium may
be diluted up to 10,000-fold before adding to the cell culture
medium to grow fish cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The disclosure may be better understood by reference to the
detailed description when considered in connection with the
accompanying drawings. The components in the figures are not
necessarily to scale, emphasis instead being placed upon
illustrating the principles of the disclosure.
[0017] FIG. 1 is a flowchart of a method for meat production by in
vitro cell culture, according to one embodiment of the present
disclosure.
[0018] FIG. 2 is a schematic representation of a method for
post-transcriptional enhancement of protein expression, according
to one embodiment of the present disclosure.
[0019] FIG. 3 is a schematic representation of a method for
post-transcriptional enhancement of collagen, type 1, alpha 1
(COL1A1) expression, according to one embodiment of the present
disclosure.
[0020] FIG. 4 is a schematic representation of a method for
post-transcriptional enhancement of collagen, type 1, alpha 2
(COL1A2) expression, according to one embodiment of the present
disclosure.
[0021] FIG. 5 is a schematic or conceptual cross-sectional view of
a bioreactor used for in vitro meat production having a solid phase
support, according to one embodiment of the present disclosure.
[0022] FIG. 6 is a schematic or conceptual cross-sectional view of
a bioreactor similar to FIG. 5 but having a second solid phase,
according to one embodiment of the present disclosure.
[0023] FIG. 7 is a chart illustrating the respective cell numbers
after treating MCF-7 cells with different concentrations (1
.mu.g/ml to 100 ng/ml) of recombinant human IGF-1 (Oryzogen). Cells
were harvested on day 10 for direct cell counting.
[0024] FIG. 8 is a chart illustrating the respective relative
fluorescence after the treatment of MCF-7 cells with 15 nM of
recombinant human IGF-1 (from 3 different suppliers), recombinant
mouse IGF-1, and recombinant fish (tuna, bream) IGF-1. Cells were
harvested on day 7 and subjected to CyQUANT Cell Proliferation
Assay.
[0025] FIG. 9 is a chart illustrating the respective relative
fluorescence after the treatment of fish swim bladder cells with 10
nM of three different clones of recombinant fish IGF-1. Cells were
harvested on day 3 and subjected to CyQUANT Cell Proliferation
Assay.
[0026] FIG. 10 is a chart illustrating the respective relative
fluorescence after the treatment of fish swim bladder cells with 1%
of three different recombinant fish IGF-1-containing yeast culture
medium. Cells were harvested on day 3 and subjected to CyQUANT Cell
Proliferation Assay.
DETAILED DESCRIPTION
[0027] Referring now to the drawings, and with specific reference
to FIG. 1, a method 10 for in vitro meat production is shown. As
used herein, "in vitro meat production" refers to a cell-based meat
production process or cell-based agriculture process in which
tissues from animals and/or plants are grown in laboratories using
cell culture techniques to manufacture meat and meat products. At a
block 12, tissue from an animal or a plant is isolated. In one
embodiment, the tissue is derived from bony fish of the class
Osteichthyes including saltwater fish such as a grouper, sea bass,
or a yellow cocker. In other embodiments, other types of animal
tissue, such as cow tissue, may be isolated. In some embodiments,
the block 12 may involve collecting organ tissue, such as a swim
bladder, from a fish and making a cell suspension. Although the
following description primarily describes tissues derived from fish
sources, it will be understood that the concepts may be applied to
tissues derived from other types of animal sources and/or plant
sources to provide other types of in vitro meat and/or animal
protein products, and vegetarian meat and/or protein products.
[0028] Many of the isolated cells are adult cells, and can be made
to proliferate continuously using various established methods in
medical research (block 14). For example, specific genes, such as
Yamanaka factors, may be used to reprogram the adult cells into
stem cells, such as induced pluripotent stem cells (iPSCs).
Alternatively, the isolated adult cells may be transformed into
continuous cell lines by telomerase reverse transcriptase
overexpression. In other embodiments, other types of cells may be
isolated such as adult stem cells and embryonic stem cells. In this
regard, it will be understood that the methods of the present
disclosure include all sources of cell lines.
[0029] In a next block 16, the cells are grown into a solid or
semi-solid structure mimicking an animal organ, such as a fish
organ, by attaching/adhering to a food-grade biocompatible scaffold
in a sterile chamber or container, such as a bioreactor. The
sterile chamber or container may be temperature controlled, and may
have inlets and outlets for introducing and removing substances
such as chemicals, nutrients, and cells. The food-grade
biocompatible scaffold becomes part of the final edible product,
and is made of plant-based or fungi-based materials such as, but
not limited to, agarose, alginate, chitosan, mycelium, and konjac
glucomannan. Alginate is a biopolymer naturally derived from brown
algae and is biocompatible. In addition, plant-based chitosan from
fungi has antibacterial properties. In some embodiments, the block
16 is carried out in the absence of antibiotics or antimicrobial
compounds in the sterile container. A block 18 involves supplying
the culture medium to the bioreactor to support cell survival and
growth. The culture medium may be a buffered solution containing
components such as, but not limited to, inorganic salts (e.g.,
calcium chloride (CaCl.sub.2)), potassium chloride (KCl), sodium
chloride (NaCl), sodium bicarbonate (NaHCO.sub.3), sodium
dihydrogen phosphate (NaH.sub.2PO.sub.4), magnesium sulfate
(MgSO.sub.4), etc.), amino acids, vitamins (e.g., thiamine,
riboflavin, folic acid, etc.), and other components such as
glucose, .beta.-mercaptoethanol, ethylenediaminetetraacetic acid
(EDTA), and sodium pyruvate. Non-limiting examples of growth media
include, but are not limited to, Leibovitz's L-15 medium, Eagle's
Minimum Essential Media (MEM), Medium 199, Dulbecco's Modified
Eagle Medium (DMEM), Ham's F12 Nutrient Mix, Ham's F10 Nutrient
Mix, MacCoy's 5A Medium, Glasgow Modified Eagle Medium (GMEM),
Iscove's Modified Dulbecco's Medium, and RPMI 1640.
[0030] According to a block 20, food-grade growth factors and
cytokines are introduced into the culture medium in the bioreactor
to support cell growth and proliferation. The growth factors and
cytokines may include, but are not limited to, insulin growth
factor 1 (IGF-1), insulin, interleukin 6 (IL-6), interleukin 6
receptor (IL-6R), interleukin 11 (IL-11), fibroblast growth factor
(FGF), epidermal growth factor (EGF), and transferrin. The growth
factors of (i) genetically same or similar species to the isolated
cells and/or (ii) genetically same genus to the isolated cells
(i.e. cells growing at block 16) are used in the present invention.
It is found that the use of growth factors of (i) genetically same
or similar species to the isolated cells and/or (ii) genetically
same genus to the isolated cells exert higher bioactivities to the
isolated cells compared to the use of growth factors and serum of
genetically distant species to the isolated cells. Due to the
enhanced compatibility, there is no need to supply a megadose of
"suboptimal" growth factors when culturing isolated cells using
growth factors of (i) genetically same or similar species and/or
(ii) genetically same genus. Higher bioactivity could also help
reducing the amount of growth factors needed in the culture medium,
shortening the culture period and improving cell quality. The cost
of the culture medium could be reduced due to the decrease in the
levels of growth factors required in the culture medium for the
stimulations of cell growth and differentiation. Furthermore, the
use of suboptimal growth factors limits the magnitude of the
maximum cellular response (growth, differentiation). In some
instances, certain responses can never be reached no matter how
much of the suboptimal growth factor is supplied. Species-specific
and/or genus-specific growth factors could help overcoming these
limits.
[0031] In some embodiments, the species is genetically similar to
the isolated cells when they are more than 90% match in DNA
sequence.
[0032] Species-specific or genus-specific growth factors can
effectively act on receptors of the isolated cells. Compared to the
conventional growth medium, which is often supplemented by high
levels of human protein growth factors and/or FBS irrespective of
the species origin of the isolated cell, the use of
species-specific or genus-specific growth factors is better
optimized. Species-specific or genus-specific variations in amino
acid sequence and post-translational modifications of the growth
factor(s) and cell receptor(s) may account for this phenomenon.
[0033] As will be discussed in much greater detail below, to
identify which species of a certain growth factor exerts the
highest bioactivity on the target isolated cells, target cells are
first seeded in complete medium (i.e. basal medium+FBS). Upon
reaching the target confluence (around 20%-70%), target cells are
treated by the growth factor of different species at a range of
concentrations (e.g. 1 pM-1 .mu.M). Target cells are kept in the
incubator until reaching the desired time point(s) for the studied
parameter (e.g. cell growth, differentiation markers, cellular
products). For example, when there are differences in cell
confluence between the treatment groups (around 2-10 days), cell
growth can be measured by trypan blue exclusion, the CyQUANT assay,
or any other appropriate cell proliferation/death assays. The
bioactivities of the growth factors of various species are compared
based on their EC50 values (half-maximal effective concentration).
For cost-effectiveness, target cells should be cultured using the
growth factor with the lowest EC50 value. However, if the aim is to
attain the shortest culturing time or the highest cell quality,
select the growth factor which triggers the highest maximum
cellular response. The optimal dose of the growth factor is defined
as the lowest concentration required to elicit the maximum cellular
response.
[0034] In some embodiments, the block 20 may involve co-culturing
bioengineered cells with the isolated cells in the absence of fetal
bovine serum (FBS). The bioengineered cells are engineered to
secrete the above growth factors and cytokines, and supply these
biomolecules to the isolated cells as needed for growth and
proliferation. As used herein, "bioengineered" cells are not
equivalent to genetically-modified cells. The bioengineered cells
have a specific gene that overexpresses one or more specific
proteins. The bioengineered cells may be fish cells, or other types
of animal cells, such as cow cells. The bioengineered cells and the
isolated cells may be genetically similar or identical species.
Also, the bioengineered g cells and the isolated cells may belong
in the same genus. As non-limiting examples, bioengineered fish
cells may be co-cultured with isolated fish cells, or bioengineered
cow cells may be co-cultured with isolated cow cells. In some
particular examples, the bioengineered cells may be chicken cells
or bird cells if chicken cells are used as the isolated cells. In
yet another particular example, the bioengineered cells may be
yellow crocker cells or other fish cells if yellow crocker cells
are used as the isolated cells. The bioengineered cells are not
present in the final meat product. The co-culturing method of the
present disclosure eliminates the need for animal-derived fetal
bovine serum (FBS) in the culture medium. Furthermore, the
co-culturing method provides a continuous supply of food-grade
specific growth factors and cytokines to the growing isolated cells
in situ, and simplifies and reduces the cost of the production
process, wherein the growth factors are (i) of genetically same or
similar species to the isolated cells and/or (ii) of the same genus
to the isolated cells. However, in other embodiments, FBS or other
serum may be used to supply growth factors, cytokines, and other
nutrients to support cell growth during the block 16.
[0035] In some embodiments, the block 20 contains recombinant
growth factors of genetically same or similar species to the
isolated cells. In yet some embodiments, the recombinant growth
factors of the same genus to the isolated cells are used. The
recombinant growth factors are introduced into the growth medium.
The use of such recombinant growth factors exerts higher
bioactivities on the isolated cells than growth factors and serum
of distant species on the isolated cells. Bacterial, yeast, insect,
mammalian, or any other appropriate protein expression systems may
be used to produce such recombinant growth factors. Protein
purification is performed by (but not limited to) affinity
chromatography, ion-exchange chromatography, size exclusion
chromatography, or a combination of these strategies.
[0036] In some embodiments, recombinant growth factors of
Epinephelus akaara (fish), which is genetically similar species to
the fish muscle cells or swim bladder cells of Epinephelus awoara
(fish), are used. The recombinant growth factors for culturing
Epinephelus awoara used are Epinephelus akaara's IGF-1, insulin
and/or transferrin. The concentration of such IGF-1 is ranged from
10 ng/ml to 100 ng/ml. The concentration of such insulin is ranged
from 1 .mu.g/ml-10 .mu.g/ml. The concentration of such transferrin
is ranged from 0.5 .mu.g/ml-5 .mu.g/ml.
[0037] Optionally, according to a block 22, protein expression in
the cells may increase the biomass yield in the resulting meat
product. As used herein, "biomass yield" refers to the amount of
digestible material (e.g., proteins) in the resulting meat product
that is available for energy production upon consumption. Again, as
an optional feature, the block 22 may involve increasing protein
expression by altering micro RNA levels in the cells, with the
manipulation of the cells being carried out prior to culturing.
Micro RNAs are endogenous, short, non-encoding single-stranded RNA
sequences involved in regulating post-transcriptional gene
expression. The block 22 may optionally involve increasing the
amount of up-regulating micro RNAs that increase protein expression
by promoting messenger RNA (mRNA) translation, and/or decreasing
the amount of down-regulating micro RNAs that decrease protein
expression by suppressing mRNA translation. The micro RNA levels
may be increased or decreased by introducing micro RNAs, micro RNA
mimics, or micro RNA inhibitors into the cells. The micro RNA
mimics have the same function as micro RNAs, but may be more stable
and efficient in modulating protein expression. In some
embodiments, electroporation may be used to introduce episomal
vectors into the cells that carry instructions to express specific
micro RNAs. Alternatively or in combination with this, an
adeno-associated virus may be used as a vehicle carrying episomal
instructions to express specific micro RNAs. Decreasing the amount
of targeted down-regulating micro RNAs may be achieved by
introducing inhibitors for the targeted micro RNAs into the cells
by transfection. It is noted here that the methods of increasing
protein expression/biomass yield according to the present
disclosure is carried out without modifying the genome of the
cells.
[0038] Turning to FIG. 2, a method for post-transcriptional
enhancement of protein expression in the cell lines is
schematically depicted. One or more up-regulating micro RNAs
(miRNAs) may be increased to increase mRNA translation and protein
production of selected proteins. Alternatively or in combination
with this, one or more down-regulating miRNAs may be blocked with
inhibitors (anti-miRNAs) to increase mRNA translation and protein
production of selected proteins.
[0039] Fish swim bladder primarily includes fibroblasts and
collagen protein. Collagen type 1 (collagen I) is a dominant
protein in the fish swim bladder, and increased expression of
collagen I in cultured fish swim bladder cells may increase biomass
yield. Collagen I in the fish swim bladder cells includes collagen,
type 1, alpha 1 (COL1A1) and collagen, type 1, alpha 2 (COL1A2).
COL1A1 and COL1A2 expression are increased by up-regulating
microRNA 21 (miR-21), such that increasing levels of miR-21
increase COL1A1 and COL1A2 production in fish swim bladder cells.
Additionally, COL1A1 and COL1A2 expression are decreased by
down-regulating microRNA 29a (miR-29a), such that decreasing levels
of miR-29a or blocking the action of miR-29a increases COL1A1 and
COL1A2 production in fish swim bladder cells. FIGS. 3-4 show
increasing COL1A1 (FIG. 3) and COL1A2 (FIG. 4) production by
increasing miR-21 levels and by blocking the action of miR-29a with
the use of inhibitors (anti-miR 29a). Increased COL1A1 and COL1A2
production results in increased biomass yield in the resulting meat
product. Similar strategies may be applied to increase relevant
protein levels in other types of animal cells.
[0040] Turning to FIG. 5, an exemplary bioreactor 30 used for
culturing the isolated cells is shown. The cells attach to and grow
on a solid phase support 32 provided by a food-grade scaffold 34
which is held in a sterile chamber 36 in the bioreactor 30. The
scaffold 34 may dictate the shape of the meat product. The
food-grade scaffold 34 is made of plant-based or fungi-based
materials such as, but not limited to, agarose, alginate, chitosan,
mycelium, and konjac glucomannan. The solid phase support 32 may be
porous so that the cells may attach to and grow on inner surfaces
of the support 32. The culture medium supplying nutrients to the
cells is introduced into the bioreactor 30 through an inlet 38, and
is emptied from the bioreactor 30 through an outlet 40.
[0041] FIG. 6 shows a bioreactor 50 similar to the bioreactor 30 of
FIG. 5, but further includes a second solid phase 52 separated from
the solid phase support 32 by a fine mesh 54. The second solid
phase 52 may contain or support the bioengineered cells that
secrete nutrients, growth factors, and cytokines for the cells
growing on the solid phase support 32 in situ, and may physically
separate the bioengineered cells from the cells on the solid phase
support 32. The second solid phase 52 is made of plant-based
materials, similar to the solid phase support 32. The mesh 54 is
permeable to nutrients, growth factors, and cytokines, but is
impermeable to cells. The bioreactor 50 of FIG. 6 allows the
co-culturing of the bioengineered cells with the growing cells. In
some embodiments, the bioreactors 30 and 50 of FIGS. 5 and 6 may be
arranged in tandem. In other embodiments, several of the
bioreactors 30, several of the bioreactors 50, or mixtures of the
bioreactors 30 and 50 may be arranged in series for scaling up the
process. The bioreactor 30 may be used mainly for biomass
production, whereas the bioreactor 50 may be used for providing
nutrients, growth factors, and cytokines to the growing cells.
[0042] The in vitro meat production method of the present
disclosure provides meat products with a simple tissue organization
of one cell type. The meat product with one cell type is easier to
make, develop, and commercialize compared to other cultured meats
having multiple cell types. Alternative embodiments of the present
disclosure provide meat products with multiple cell types.
Furthermore, Applicant has discovered a strategy to increase
biomass/protein production by altering micro RNA levels or activity
in the growing cells. In one example, two key micro RNAs (miR-21
and miR-29a) are targeted to increase the levels of the dominant
protein (collagen I) found in fish swim bladder cells. As far as
the Applicant is aware, alteration of micro RNA levels or activity
to achieve an increased protein/biomass yield in cultured meat
products has not been used by others in the field of cultured meat
development. Targeting micro RNAs for increased protein production
may cause less stress to the cells than known knock-in or knock-out
methods. Bio-engineered cells are co-cultured with the growing
animal cells to supply the growing fish cells with food-grade
growth factors and cytokines for cell growth and proliferation in
situ, reducing or eliminating the need for animal-derived FBS in
the culture medium. The co-culturing technique simplifies the
production process and reduces production costs.
[0043] Furthermore, the nutrients of the cultivated meat product
may be customized to generate a healthier food product. For
example, the cultured meat product may be customized according to
diet recommendations from a dietician or from a personal genomic
test. Healthy nutrients such as high-density cholesterol,
polyunsaturated fatty acids, and monounsaturated fatty acids in the
meat product may be enriched by culturing the cells in specific
conditions. Alternatively, or in combination with this, nutrients
known to be damaging to health such as low-density cholesterol and
saturated fatty acids may be reduced by culturing the cells in
specific conditions. Micronutrients, such as vitamins and minerals,
may also be enhanced. Nutrient customization of the cultivated meat
products may be achieved in various ways such as, but not limited
to, 1) tailoring the nutrients fed to the growing cells during cell
culture, and/or 2) controlling the proportions of layering
scaffolds with different cells.
[0044] The production of the cultivated food product is under a
clean, sterile and highly controlled process. Thus, undesirable
degradation by microorganisms such as bacteria or fungi of the
nutrients in the food product is minimized. Undesirable tastes and
smells from the breakdown of nutrients by microorganisms are also
minimized. This property of cultivated food enables new uses in
cooking and helps creates novel recipes. One such application of
cultivated food is cultivated fish maw derived from fish swim
bladders. Traditional fish maw has an undesirable fishy taste and
smell due to the degradation of amine by bacteria in the production
process. This undesirable property limits the food ingredient to
savory dishes served hot or warm. Cultivated fish maw produced from
cell culture technology does not have an undesirable fishy taste
and smell. In addition to hot and savory dishes, cultivated fish
maw can be used in sweet dishes, as a dessert or in a ready-to-eat
format served at chilled or at ambient temperature.
[0045] Identification of Species-Specific or Genus-Specific Growth
Factor
[0046] The method of identifying species-specific or genus-specific
growth factors will be discussed in detail here. It includes two
major steps, which are the cell growth stimulation step and the
measuring cell growth step.
[0047] Cell Growth Stimulation Step
[0048] The MCF-7 human epithelial cell line is cultured in DMEM/F12
complete medium DMEM/F12, 10% FBS inside a humidified incubator
(34.degree. C.; 5% CO.sub.2; 95% air). Split cells at a ratio of
1:4 to 1:8 for routine maintenance.
[0049] Upon reaching about 80% confluence, detach cells by
trypsin/EDTA. To study the effect of growth factors on cell growth,
cells are seeded at a density of 3.times.10.sup.4 cells/cm.sup.2 in
complete medium onto 24-well (if cell growth is measured by cell
counting) or 96-well plates (if cell growth is measured by the
CyQUANT Cell Proliferation Assay Kit). Return the cells to the
incubator.
[0050] After 24 hours, remove the medium. Pre-adapt cells to
serum-free conditions by adding serum-free medium DMEM/F12, 0.1%
human serum albumin. Keep the cells in this medium for at least 16
hours inside the incubator.
[0051] Prepare growth factors (e.g. IGF-1) at 10.times. working
concentrations in serum-free medium for each species/genius, which
growth-stimulating effect to be examined (e.g. recombinant human
IGF-1, human IGF-1-LR3, mouse IGF-1, bream IGF-1 and tuna IGF-1).
Add the 10.times. growth factors into the wells such that cells
will be treated by 1.times. growth factors (e.g. add 50 .mu.l
10.times. growth factor to well containing 450 .mu.l serum-free
medium) (e.g. 1 pM-1 .mu.M). Return the cells to the incubator.
[0052] Observe the cells daily under a microscope for signs of cell
growth. When there are obvious differences in terms of cell
confluence between the treatment groups (usually detected between
day 2-day 10), quantify cell growth either by cell counting, the
CyQUANT Cell Proliferation Assay, or any other cell
proliferation/death assays.
[0053] Measuring Cell Growth Step
[0054] There are two ways to measure cell growth, namely, trypan
blue exclusion and CyQUANT Cell Proliferation Assay Kit.
[0055] Trypan Blue Exclusion
[0056] Cells should have been treated in 24-well plates. Aspirate
the culture medium and detach cells by trypsin-EDTA.
[0057] Stop trypsin activity by adding 1 volume of the complete
medium into the well. Ensure that all cells are detached by
pipetting 3-5 times inside the well.
[0058] Collect the cell suspension into 1.5 ml tubes. Pellet the
cells by centrifuging the tubes at 400.times.g for 5 minutes.
[0059] Remove the supernatant without disturbing the cell pellet.
Resuspend the cell pellet in 200 .mu.l DMEM/F12 basal medium.
[0060] Mix 10 .mu.l of the cell suspension with 10 .mu.l of 0.4%
Trypan Blue solution
[0061] After 2 minutes, add 10 .mu.l of the cell/trypan blue
mixture to each chamber of a Countess II FL Disposable Slide or a
hemocytometer. If using the Countess II FL system, insert the slide
into the slide holder of a Countess II FL Automated Cell Counter
and determine the cell concentration and % viability. If using a
hemocytometer, count cells with a microscope.
[0062] Calculate the number of cells in each treatment group. The
number of viable cells per well equals to viable cell concentration
(cells/ml).
[0063] CyQUANT Cell Proliferation Assay Kit
[0064] The CyQUANT Cell Proliferation Assay Kit quantifies cell
growth by measuring the nucleic acid content in samples. Cells
should have been seeded onto 96-well plates, preferably in
triplicate wells per treatment group.
[0065] Remove the culture medium as much as possible by a
multichannel pipette. Avoid scratching the well bottom with the
pipette tip.
[0066] Freeze the plate in a -80.degree. C. freezer. The plate may
be stored at -80.degree. C. for up to 4 weeks.
[0067] Thaw the plate and assay kit reagents at room
temperature.
[0068] Mix the kit reagents according to Table 1 for each well.
TABLE-US-00001 TABLE 1 Volume per well Components (.mu.l) Cell
lysis buffer stock solution 10 .mu.l Autoclaved MilliQ water 189.5
.mu.l CyQUANT GR stock solution 0.5 .mu.l Total = 200 .mu.l
[0069] When the plate has completely thawed, add 200 .mu.l of the
CyQUANT GR dye/lysis buffer mixture to each sample well and to
three empty wells (blank). Incubate the plate at room temperature
for 5 minutes, protected from light.
[0070] Using a multichannel pipette, transfer 160 .mu.l from each
well of the 96-well plate to the corresponding well of a black
96-well plate.
[0071] Measure the sample fluorescence using a fluorescence
microplate reader (e.g. Molecular Devices SpectraMax iD5). Set the
excitation and emission wavelengths at 480 nm and 520 nm
respectively.
[0072] Average the blank wells fluorescence readings. Subtract this
average reading from all sample fluorescence readings to correct
for background fluorescence.
[0073] Calculate the mean corrected fluorescence for the vehicle
group. Express the treatment group fluorescence readings as fold of
control (FOC) by dividing the sample readings (i.e. IGF-1 groups)
by the mean vehicle reading.
Example 1
[0074] IGF-1 Stimulated the Growth of MCF-7 Cells in a
Dose-Dependent Manner
[0075] To verify that IGF-1 stimulates the growth of human MCF-7
cells, cells were treated with increasing doses of human IGF-1 (0
.mu.g/ml-100 ng/ml) for 10 days and processed for cell counting. As
seen in FIG. 7, while 1-100 .mu.g/ml IGF-1 did not enhance the cell
number, further increase of IGF-1 concentration (1-100 ng/ml)
promoted cell growth in a dose-dependent manner. Hence, MCF-7 cells
are suitable for evaluating the growth-stimulating activity of
IGF-1.
Example 2
[0076] Human IGF-1, but not the Mouse or Fish IGF-1, Promoted the
Growth of Human MCF-7 Cells
[0077] To investigate whether the growth-stimulating activity of
IGF-1 depends on its species origin, we treated MCF-7 cells with
1.5 nM recombinant IGF-1 of various species, i.e. human, mouse, and
fish (bream, tuna). After 7 days, cell growth was assessed by the
CyQUANT Assay (FIG. 8). While human IGF-1 obtained from multiple
sources consistently increased MCF-7 cell growth (.about.50-100%
increase), mouse and fish IGF-1 did not (FIG. 8). These findings
suggest that human IGF-1, being the same species as the human MCF-7
cells, is more effective than fish and mouse IGF-1 in promoting
cell growth.
Example 3
[0078] Fish IGF-1 expressed by recombinant yeast cells promoted
growth of fish swim bladder cells.
[0079] To investigate the effects of growth factors produced by
microorganisms such as yeasts on the growth of animal cells, fish
IGF-1 was produced by and obtained from a recombinant yeast strain
that was genetically engineered to carry and express a fish IGF-1
gene, and then added to the culture medium for growing fish swim
bladder cells. FIG. 9 shows a chart illustrating the respective
relative fluorescence after the treatment of fish swim bladder
cells by 10 nM of three different clones of recombinant fish IGF-1,
each of which has a different nucleotide sequence from each other
and the native gene of fish IGF-1. For example, the cells were
harvested on day 3 and subjected to CyQUANT Cell Proliferation
Assay.
[0080] FIG. 10 is a chart illustrating the respective relative
fluorescence after the treatment of fish swim bladder cells by 1%
(v/v) of three different batches of yeast culture medium, each of
which contains exclusively one of the clones of recombinant fish
IGF-1 as mentioned in the preceding paragraph. In this example, the
three batches of IGF-1-containing yeast medium were collected,
centrifuged and filtered, and the resulting supernatants were added
directly to the fish swim bladder cells. The cultured cells were
harvested on day 3 and subjected to CyQUANT Cell Proliferation
Assay.
[0081] The results in FIGS. 9 and 10 suggest that the addition of
fish IFG-1 obtained from recombinant yeast culture to the culture
medium of fish swim bladder cells enhanced the growth of the swim
bladder cells. A skilled person in the art would appreciate that
apart from IFG-1, other growth factors and cytokines can also be
used in connection with these embodiments and other embodiments of
this invention, including but not limited to insulin, interleukin 6
(IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11),
fibroblast growth factor (FGF), epidermal growth factor (EGF), and
transferrin, and cells from prokaryotic organisms or eukaryotic
organisms can be used as the recombinant cells for producing the
target growth factors or cytokines. Apart from yeast, other types
of microorganisms such as bacteria, archaea, fungi, algae,
protozoa, and viruses, and other types of cells such as plant
cells, insect cells and mammalian cells may also be used. A skilled
person in the art would also appreciate that the target isolated
cells to be treated with such exogenous growth factors or cytokines
are not limited to fish cells, and that the gene of the growth
factors or cytokines to be introduced into the recombinant cells
(such as yeast) as an expression system by way of genetic
engineering shall be preferably (i) of genetically same or similar
species to the target cells and/or (ii) of genetically same genus
to the target cells.
[0082] In one embodiment, the concentration of recombinant growth
factors or cytokines to be added to the culture medium of the
target isolated cells are in the range of 0.1%-1% (v/v) or 1 nM-10
nM.
[0083] The present invention shows that it is more effective to
apply growth factors and albumin of (i) genetically same or similar
species or (ii) same genus as the cultured cell type. The usage of
these growth factors or protein factors may be decreased while
achieving the same growth rate. Species-specific growth factors
and/or genus-specific growth factors represent a promising
direction to reduce media cost especially during large-scale cell
production for cultivated meat and other applications using the
cultivated cell mass. Using more bioactive growth factors can also
decrease processing times and improve the quality (e.g. texture,
taste, nutritional value) of cultivated meat or cell mass.
[0084] The above description is illustrative and is not
restrictive. Many variations of embodiments may become apparent to
those skilled in the art upon review of the disclosure. The scope
embodiments should, therefore, be determined not with reference to
the above description, but instead should be determined with
reference to the pending claims along with their full scope or
equivalents.
[0085] One or more features from any embodiment may be combined
with one or more features of any other embodiment without departing
from the scope embodiments. A recitation of "a", "an" or "the" is
intended to mean "one or more" unless specifically indicated to the
contrary. Recitation of "and/or" is intended to represent the most
inclusive sense of the term unless specifically indicated to the
contrary.
[0086] While the present disclosure may be embodied in many
different forms, the drawings and discussion are presented with the
understanding that the present disclosure is an exemplification of
the principles of one or more inventions and is not intended to
limit any one embodiment to the embodiments illustrated.
[0087] The disclosure, in its broader aspects, is therefore not
limited to the specific details, representative system and methods,
and illustrative examples shown and described above. Various
modifications and variations may be made to the above specification
without departing from the scope or spirit of the present
disclosure, and it is intended that the present disclosure covers
all such modifications and variations provided they come within the
scope of the following claims and their equivalents.
Exemplary Protocols
[0088] A. Development of a Fish Bladder Cell Line
[0089] Obtain a healthy yellow crocker, sea bass or fish of a
similar category from a local fish market.
[0090] Keep the fish on ice until cell isolation.
[0091] Immerse the fish in 10% bleach.
[0092] Remove swim bladder from the fish under aseptic
condition.
[0093] Wash the organ one or more times in hypochlorous acid.
[0094] Wash the organ one or more times in antibiotic medium
(Leibovitz's L-15 or DMEM or EMEM with 400 IU/ml, penicillin, 400
.mu.g/ml streptomycin).
[0095] After washing, cut the organ into small pieces (2-3
mm.sup.3).
[0096] Transfer the cut organ to a centrifuge tube containing 0.25%
trypsin-EDTA in PBS.
[0097] Incubate at room temperature with continuous shaking for 1
hour.
[0098] Filter the supernatant with a 100 pm mesh to remove
undigested tissue.
[0099] Centrifuge the filtrate at 200 g for 5 minutes.
[0100] Resuspend the cell pellet with complete medium (Leibovitz's
L-15 or DMEM or EMEM with 200 IU/ml, penicillin, 200 .mu.g/ml
streptomycin, 10% fetal bovine serum).
[0101] Seed the cell into a T25 flask.
[0102] Incubate at 24-28.degree. C.
[0103] Remove cells that are not attached to the tissue culture
flask the next day.
[0104] Replace half of the medium with fresh medium every 2-3
days.
[0105] The cells are considered established when a complete
monolayer is formed and the established cells are ready for
subculture.
[0106] B. Development of a Fish Bladder Cell Line by Tissue
Explant
[0107] Obtain a healthy yellow crocker, sea bass, or fish of a
similar category from a local fish market.
[0108] Keep the fish on ice until cell isolation.
[0109] Immerse the fish in 10% bleach.
[0110] Remove swim bladder from the fish under aseptic condition.
[0111] Wash the organ one or more times in hypochlorous acid.
[0112] Wash the organ one or more times in antibiotic medium
(Leibovitz's L-15 or DMEM or EMEM with 400 IU/ml, penicillin, 400
.mu.g/ml streptomycin). [0113] After washing, cut the organ into
small pieces (1-2 mm.sup.3). [0114] Place organ pieces into a 24
well plate individually containing complete medium (Leibovitz's
L-15 or DMEM or EMEM with 200 IU/ml, penicillin, 200 .mu.g/ml
streptomycin, 10% fetal bovine serum).
[0115] Incubate at 24-28.degree. C.
[0116] Replace half of the medium with fresh medium every 2-3 days
without disturbing the tissue explant.
[0117] Incubate the tissue explant until adherent cells are
observed.
[0118] Remove tissue explant.
[0119] The cells are considered established when a complete
monolayer is formed and the established cells are ready for
subculture.
[0120] C. Development of a Fish Muscle Cell Line
[0121] Obtain a healthy grouper, cod, sole, halibut, flounder, or
fish of a similar category from a local fish market.
[0122] Keep the fish on ice until cell isolation.
[0123] Immerse the fish in 10% bleach.
[0124] Remove muscle from the fish under aseptic condition.
[0125] Wash the tissue one or more times in hypochlorous acid.
[0126] Wash the tissue one or more times in antibiotic medium
(Leibovitz's L-15 or DMEM or EMEM with 400 IU/ml, penicillin, 400
.mu.g/ml streptomycin).
[0127] After washing, cut the tissue into small pieces (2-3
mm.sup.3).
[0128] Transfer the cut tissue to a centrifuge tube containing
collagenase and dispase in PBS.
[0129] Incubate at room temperature with continuous shaking for 1
hour.
[0130] Filter the supernatant with a 100 pm mesh to remove
undigested tissue.
[0131] Centrifuge the filtrate at 200 g for 5 minutes.
[0132] Resuspend the cell pellet with complete medium (Leibovitz's
L-15 or DMEM or EMEM with 200 IU/ml, penicillin, 200 .mu.g/ml
streptomycin, 10% fetal bovine serum).
[0133] Seed the cell into a T25 flask.
[0134] Incubate at 24-28.degree. C.
[0135] Remove cells that are not attached to the tissue culture
flask the next day.
[0136] Replace half of the medium with fresh medium every 2-3
days.
[0137] The cells are considered established when a complete
monolayer is formed and the established cells are ready for
subculture.
[0138] D. Development of a Fish Muscle Cell Line from Tissue
Explant
[0139] Obtain a healthy grouper, cod, sole, halibut, flounder, or
fish of a similar category from a local fish market.
[0140] Keep the fish on ice until cell isolation.
[0141] Immerse the fish in 10% bleach.
[0142] Remove muscle from the fish under aseptic condition.
[0143] Wash the tissue one or more times in hypochlorous acid.
[0144] Wash the tissue one or more times in antibiotic medium
(Leibovitz's L-15 or DMEM or EMEM with 400 IU/ml, penicillin, 400
.mu.g/ml streptomycin).
[0145] After washing, cut the muscle into small pieces (1-2
mm.sup.3).
[0146] Place muscle pieces into a 24 well plate individually
containing complete medium (Leibovitz's L-15 or DMEM or EMEM with
200 IU/ml, penicillin, 200 .mu.g/ml streptomycin, 10% fetal bovine
serum).
[0147] Incubate at 24-28.degree. C.
[0148] Replace half of the medium with fresh medium every 2-3 days
without disturbing the tissue explant.
[0149] Incubate the tissue explant until adherent cells are
observed.
[0150] Remove tissue explant.
[0151] The cells are considered established when a complete
monolayer is formed and the established cells are ready for
subculture.
[0152] E. Adult Stem cell isolation and culture
[0153] Obtain a healthy grouper, cod, sole, halibut, flounder or
fish 6 months or younger of similar category from a local fish
market.
[0154] Keep the fish on ice until cell isolation.
[0155] Immerse the fish in 10% bleach.
[0156] Remove muscle from the fish under aseptic conditions.
[0157] Wash the tissue one or more times in hypochlorous acid.
[0158] Wash the tissue one or more times in antibiotic medium
(Leibovitz's L-15 or DMEM or EMEM with 400 IU/ml, penicillin, 400
.mu.g/ml streptomycin).
[0159] After washing, cut the tissue into small pieces (2-3
mm.sup.3).
[0160] Transfer the cut tissue to a centrifuge tube containing
collagenase and dispase in PBS.
[0161] Incubate at room temperature with continuous shaking for 1
hour.
[0162] Filter the supernatant with a 100 pm mesh to remove
undigested tissue.
[0163] Centrifuge the filtrate at 200 g for 5 minutes.
[0164] Resuspend the cell pellet with complete medium (Leibovitz's
L-15 or DMEM or EMEM with 200 IU/ml, penicillin, 200 .mu.g/ml
streptomycin, 10% fetal bovine serum, 100 ng/ml basic fibroblast
growth factor).
[0165] Plate the cells on an uncoated plate for 1 hour at
24-28.degree. C.
[0166] Harvest the supernatant and place on a plate coated with
laminin, gelatin, Matrigel or similar matrix.
[0167] Incubate at 24-28.degree. C.
[0168] After 24 hours, wash away any loosely attached and
non-adherent cells.
[0169] Replace medium every day with complete medium (Leibovitz's
L-15 or DMEM or EMEM with 200 IU/ml, penicillin, 200 .mu.g/ml
streptomycin, 10% fetal bovine serum, 100 ng/ml basic fibroblast
growth factor).
[0170] F. Generating and Culturing iPSC
[0171] 2-4 days before transfection, plate cells in complete medium
(L15 with 10% FBS) in a tissue culture flask. Cells should be
approximately 75-90% confluent on the day of transfection (Day
0).
[0172] Aspirate the medium from gelatin-coated 6-well plates and
replace them with 2 mL of fresh complete medium per well. Place the
coated plates at 37.degree. C. until ready for use.
[0173] Thaw the Epi5.TM. vectors at 37.degree. C. and place them on
wet ice until ready for use. Before use, briefly centrifuge the
thawed vectors to collect them at the bottom of the tube.
[0174] Wash the cells in PBS.
[0175] Add 3 mL of 0.05% Trypsin/EDTA to the culture flask
containing the cells.
[0176] Incubate the flask at room temperature for 3 minutes.
[0177] Add 5-8 mL of complete medium to each flask. Carefully
transfer cells into an empty, sterile 15 mL conical tube.
[0178] Check the viability by trypan blue dye exclusion cell
viability assay
[0179] Centrifuge the cells at 200 g for 2 min.
[0180] Carefully aspirate most of the supernatant and resuspend
with complete medium.
[0181] Seed cells on gelatin-coated dishes plate 50,000 to 100,000
cells per well into a 6-well plate at 30-60% confluence in 2 mL
complete medium and Incubate overnight at 24-28.degree. C.
[0182] Prewarm Opti-MEM/Reduced-Serum Medium to room temperature
and prepare Tube A and Tube B as described below.
[0183] Add 1.24 each of the two Epi5.TM. Reprogramming Vector mixes
(2.44 total) to 1184 Opti-MEM medium in a 1.5 mL microcentrifuge
tube labeled Tube A. Add 4.8 .mu.L of P3000.TM. Reagent and mix
well.
[0184] Dilute 3.64 Lipofectamine 3000 reagent in 1214 prewarmed
Opti-MEM medium in a 1.5 mL microcentrifuge tube labeled Tube
B.
[0185] To prepare a transfection master mix, add the contents of
Tube A to Tube B and mix well.
[0186] Incubate the transfection master mix for 5 minutes at room
temperature.
[0187] Mix one more time and add the entire 2504 of transfection
master mix to each well.
[0188] Incubate overnight at 24-28.degree. C.
[0189] 24 hours post-transfection, aspirate the medium from the
plates. Add 2 mL N2B27 Medium (L15 with IX N-2 supplement, IX B27
supplement, 100 ng/mL bFGF to each well.
[0190] Change the N2B27 Medium every day for a total of 14 days by
replacing the spent medium with 2 mL N2B27 Medium.
[0191] Aspirate the spent N2B27 Medium on Day 14 and replace it
with a complete medium. Resume medium changes every day at 2 mL per
well.
[0192] Observe the plates every other day under a microscope for
the emergence of cell clumps, indicative of transformed cells.
Within 15 to 21 days post-transfection, the iPSC colonies will grow
to an appropriate size for transfer.
[0193] Colonies are distinct by Day 21 and can be picked for
further culture and expansion.
[0194] G. Method for Subculturing Cells
[0195] Remove and discard the culture medium.
[0196] Briefly rinse the cell PBS to remove all traces of serum
which contains trypsin inhibitor.
[0197] Add 2-3 mL of 0.25% Trypsin-EDTA solution to the flask.
[0198] Incubate at room temperature for 1 min.
[0199] Add 5-8 mL of complete growth medium.
[0200] Aspirate cells by gently pipetting.
[0201] Add appropriate aliquots of the cell suspension to new
culture flasks at a subcultivation ratio of 1:2 to 1:3.
[0202] Incubate at 24-28.degree. C.
[0203] H. Adaption to Suspension Culture
[0204] Passage monolayer culture at a frequency appropriate for the
cell in question by trypsinization.
[0205] At each passage, wash cell monolayer with PBS and overlay
with 0.25% trypsin.
[0206] Incubate at room temperature for 5 min.
[0207] Inactivate the enzyme with a complete medium.
[0208] Harvest the cell suspension and check the viability by
trypan blue dye exclusion cell viability assay.
[0209] Seed the cell suspension into another culture flask.
[0210] Repeat passaging until the viability of the suspended cells
is equal or more than 90%.
[0211] Establish a suspension culture with 50 ml complete medium in
a spinner or shaker flask at a cell density of 0.1-0.5
million/ml.
[0212] Incubate the spinner or shaker flask suspension cultures in
a CO.sub.2 incubator under the same conditions of temperature,
humidity, and atmosphere optimal for monolayer cultures.
[0213] Adjust the cell density to 0.1-0.5 million/ml with fresh
medium every 2-3 days.
[0214] Check the viability by trypan blue dye exclusion cell
viability assay.
[0215] Establish multiple parallel cultures at cell density that
promote health cell growth.
[0216] Increase cell density gradually to 1 million/ml using part
of the culture.
[0217] If increasing cell density leads to cell death, discard the
high-density culture.
[0218] Restart high-density adaption using cell form step 12.
[0219] Scale up to a 3 L bioreactor when cells are adapted to grow
in suspension.
[0220] i. Adaption to Serum-Free Medium (Plant Hydrolysate)
[0221] Culture cells in DMEM/F12 complete medium (1:1 mixture of
DMEM medium and Ham's F12 medium, 2-4 mM glutamine, 10% FBS).
[0222] Prepare serum-free medium (1:1 mixture of DMEM medium and
Ham's F12 medium, 2-4 mM glutamine, 20% plant hydrolysate e.g. soy,
cottonseed, rapeseed, wheat, yeast or equivalent).
[0223] When cells reach confluence, replace medium with adaption
medium I (40% fresh complete medium, 40% conditioned media from the
passage before, 20% serum-free medium).
[0224] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days.
[0225] If adaption leads to cell death, discard the culture and
repeat step 3.
[0226] When cells reach confluence, replace medium with adaption
medium II (30% fresh complete medium, 30% conditioned media from
the cells in step 1, 40% serum-free medium).
[0227] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days.
[0228] If adaption leads to cell death, discard the culture and
repeat step 6.
[0229] When cells reach confluence, replace medium with adaption
medium III (20% fresh complete medium, 20% conditioned media from
the cells in step 1, 60% serum-free medium).
[0230] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days
[0231] If adaption leads to cell death, discard the culture and
repeat step 9.
[0232] When cells reach confluence, replace medium with adaption
medium IV (10% fresh complete medium, 10% conditioned media from
the cells in step 1, 80% serum-free medium).
[0233] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days
[0234] If adaption leads to cell death, discard the culture and
repeat step 12.
[0235] When cells reach confluence, replace medium with serum-free
medium.
[0236] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days.
[0237] If adaption leads to cell death, discard the culture and
repeat step 15.
[0238] The serum-free medium usage can be increased more gradually
in each step, i.e. an increase of 20% or less in each step.
[0239] J. Adaption to serum-free medium (chemically defined)
[0240] Culture cells in DMEM/F12 complete medium (1:1 mixture of
DMEM medium and Ham's F12 medium, 2-4 mM glutamine, 10% FBS).
[0241] Prepare serum free medium (1:1 mixture of DMEM medium and
Ham's F12 medium, 2-4 mM glutamine, ascorbic acid 2-phosphate
65-130 ug/ml, NaHCO.sub.3550-1100 ug/ml, sodium selenite 14-28
ng/ml, insulin 19-38 ug/ml, transferrin 11-22 ug/ml, FGF-2 100-200
ng/ml, TGF-beta 2-4 ng/ml).
[0242] When cells reach confluence, replace medium with adaption
medium I (40% fresh complete medium, 40% conditioned media from the
passage before, 20% serum-free medium).
[0243] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days
[0244] If adaption leads to cell death, discard the culture and
repeat step 3
[0245] When cells reach confluence, replace medium with adaption
medium II (30% fresh complete medium, 30% conditioned media from
the cells in step 1, 40% serum-free medium).
[0246] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days.
[0247] If adaption leads to cell death, discard the culture and
repeat step 6
[0248] When cells reach confluence, replace medium with adaption
medium III (20% fresh complete medium, 20% conditioned media from
the cells in step 1, 60% serum-free medium).
[0249] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days.
[0250] If adaption leads to cell death, discard the culture and
repeat step 9.
[0251] When cells reach confluence, replace medium with adaption
medium IV (10% fresh complete medium, 10% conditioned media from
the cells in step 1, 80% serum-free medium).
[0252] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days.
[0253] If adaption leads to cell death, discard the culture and
repeat step 12.
[0254] When cells reach confluence, replace medium with serum-free
medium.
[0255] Check the viability by trypan blue dye exclusion cell
viability assay every 2-3 days.
[0256] If adaption leads to cell death, discard the culture and
repeat step 15.
[0257] The serum-free medium usage can be increased more gradually
in each step. For example, an increase of 20% or less in each
step.
[0258] K. Post-Transcriptional Enhancement of Protein
Expression
[0259] Culture cells in complete medium (Leibovitz's L-15 or DMEM
or EMEM with 200 IU/ml, penicillin, 200 .mu.g/ml streptomycin, 10%
fetal bovine serum), or serum-free medium (DMEM/F12 with plant
hydrolysate or chemically defined compounds).
[0260] Remove and discard the culture medium.
[0261] Briefly rinse the cell PBS to remove all traces of serum
which contains trypsin inhibitor.
[0262] Add 2-3 mL of 0.25% Trypsin-EDTA solution to the flask.
[0263] Incubate at room temperature for 1 min.
[0264] Aspirate cells by gently pipetting.
[0265] Centrifuge cell at 200 g for 2 min.
[0266] Resuspend cells in complete medium or serum-free medium.
[0267] Add 0.5 million cells to each well of a 6-well plate.
[0268] Incubate at 24-28.degree. C. overnight.
[0269] Transfect micro RNA oligonucleotides (miR-21, miR-29a,
miR-21 mimic, miR-29a mimic, anti-miR-21, anti-miR-29a, or
equivalent) into the cell using polyethylenimine, liposome,
electroporation, or other methods.
[0270] Incubate at 24-28.degree. C. overnight.
[0271] Transfer the cells to a multi-layer flask, spinner flask or
shaker flask in a CO.sub.2 incubator under the same conditions of
temperature, humidity, and atmosphere optimal culture
[0272] L. Scaffolding for Cell Culture (Konjac+Gum)
[0273] Boil water with a few pieces of saffron until the color
becomes pale yellow.
[0274] Remove the saffron and rest the solution until warm.
[0275] Prepare all Dry Ingredient
[0276] Konjac-0.5-5%, preferably 3%
[0277] Baking soda--0.3-3%, preferably 2%
[0278] Perfected Xanthan Gum--0.2-2%, preferably 1.5%
[0279] Measure 100 ml of saffron solution.
[0280] Add Baking soda, Xanthan Gum sequentially. Stir the mixture
well after adding each ingredient.
[0281] Add Konjac by sprinkling little by little on top of the
solution. Keep stirring. The solution should become mushy.
[0282] Spread the konjac mixture into mold with approximately 1-15
mm thickness.
[0283] Cover the mold with the lid and rest under room temperature
for more than 30 min.
[0284] Put the mold in 4.degree. C. fridge for 4 hours.
[0285] Steam the mold under low heat for 40 minutes.
[0286] Rest the mold under room temperature for 2 hours.
[0287] Dehydrate the scaffold at 45-55.degree. C. for 15
minutes.
[0288] M. Scaffolding for Cell Culture (Alginate+Glutinous Rice
Flour)
[0289] Weigh 0.1-2 g (0.1-2%), preferably about 1 g (1%) Sodium
Alginate.
[0290] Add 100 ml water into the blender.
[0291] Add the Alginate powder into the blender and blend the
mixture until dissolved.
[0292] Cover the container with plastic film and put the Alginate
solution into the refrigerator overnight to eliminate the gas
bubbles.
[0293] Weigh 1-10 g (1-10%), preferably about 5 g (5%) Glutinous
Rice Flour and put in a mold.
[0294] Add Alginate solution into the mold with approximately 1-15
mm thickness.
[0295] Stir the mixture until all flour dissolves.
[0296] Steam the mixture under low heat for 30 min until the shape
is set.
[0297] Cover the mold with the lid and rest under room temperature
for 30 min.
[0298] Weigh 1% Calcium Lactate and stir to dissolve in water.
[0299] Immerse the scaffold with 1% Calcium Lactate solution for at
least 2.5 hours to allow the formation of the membrane around the
scaffold.
* * * * *