U.S. patent application number 15/093618 was filed with the patent office on 2016-08-11 for dried food products formed from cultured muscle cells.
The applicant listed for this patent is Francoise Suzanne MARGA. Invention is credited to Francoise Suzanne MARGA.
Application Number | 20160227831 15/093618 |
Document ID | / |
Family ID | 53753710 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160227831 |
Kind Code |
A1 |
MARGA; Francoise Suzanne |
August 11, 2016 |
DRIED FOOD PRODUCTS FORMED FROM CULTURED MUSCLE CELLS
Abstract
Dehydrated, edible, high-protein food products formed of
cultured muscle cells that are combined (e.g., mixed) with a
hydrogel (e.g., a plant-derived polysaccharide) are described.
These food products may be formed into a chip (e.g., snack chip),
that has a protein content of greater than 50%. One or more
flavorants may also be included.
Inventors: |
MARGA; Francoise Suzanne;
(Brooklyn, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MARGA; Francoise Suzanne |
Brooklyn |
NY |
US |
|
|
Family ID: |
53753710 |
Appl. No.: |
15/093618 |
Filed: |
April 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14615354 |
Feb 5, 2015 |
9332779 |
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15093618 |
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61936064 |
Feb 5, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 13/422 20160801;
A23L 13/00 20160801; A23L 13/428 20160801; A23P 10/00 20160801;
A23V 2002/00 20130101 |
International
Class: |
A23L 1/31 20060101
A23L001/31; A23L 1/0524 20060101 A23L001/0524 |
Claims
1. An edible, dehydrated food product, the food product comprising:
cultured animal muscle cells, the cultured animal muscle cells
combined with a plant-derived hydrogel; wherein the cultured animal
muscle cells and plant-derived hydrogel are formed as a dehydrated
sheet of material.
2. The food product of claim 1, further comprising a flavorant.
3. The food product of claim 1, wherein the cultured animal muscle
cells and plant-derived hydrogel are distributed throughout the
sheet of material.
4. The food product of claim 1, wherein dehydrated cultured animal
muscle cells having a diameter of between about 5 to 30 .mu.m are
distributed throughout the sheet of material.
5. The food product of claim 1, wherein the cultured animal muscle
cells are derived from one or more of: beef, veal, pork, chicken,
or fish.
6. The food product of claim 1, wherein the cultured animal muscle
cells comprise one or more of: skeletal myocytes, satellite cells,
smooth myocytes, and cardiac myocytes.
7. The food product of claim 1, wherein the plant-derived hydrogel
comprises pectin.
8. The food product of claim 1, wherein the plant-derived hydrogel
comprises a low methyl (LM) esterified pectin.
9. An edible, dehydrated food product configured as a snack chip,
the food product comprising: an edible body formed as a sheet of
material comprising dehydrated cultured animal muscle cells and a
plant-derived polysaccharide; and a flavorant, wherein the cultured
animal muscle cells and plant-derived polysaccharides are
distributed throughout the sheet of material.
10. The food product of claim 9, wherein the edible body has a
diameter that is more than ten times the thickness.
11. The food product of claim 9, wherein the cultured animal muscle
cells are derived from one or more of: beef, veal, pork, chicken,
or fish.
12. The food product of claim 9, wherein the cultured animal muscle
cells comprise one or more of: skeletal myocytes, satellite cells,
smooth myocytes, and cardiac myocytes.
13. The food product of claim 9, wherein the plant-derived hydrogel
comprises pectin.
14. The food product of claim 9, wherein the plant-derived
polysaccharide comprises a low methyl (LM) esterified pectin.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 14/615,354, filed on Feb. 5, 2015, titled
DRIED FOOD PRODUCTS FORMED FROM CULTURED MUSCLE CELLS, Publication
No. US-2015-0216216-A1, which claims priority to U.S. Provisional
Patent Application No. 61/936,064, filed on Feb. 5, 2014, titled
"DRIED FOOD PRODUCTS FORMED FROM CULTURED MUSCLE CELLS," each of
which is herein incorporated by reference in its entirety.
[0002] This patent application may also be related to one or more
(or all) of: U.S. patent application Ser. No. 14/486,850, filed on
Sep. 15, 2014, titled "EDIBLE AND ANIMAL-PRODUCT-FREE MICROCARRIERS
FOR ENGINEERED MEAT;" U.S. patent application Ser. No. 14/092,801,
filed on Nov. 27, 2013, titled "ENGINEERED COMESTIBLE MEAT," which
is a continuation of U.S. patent application Ser. No. 13/558,928,
filed Jul. 26, 2012, titled "ENGINEERED COMESTIBLE MEAT," now U.S.
Pat. No. 8,703,216, and PCT Application No. PCT/US2013/058684,
filed on Sep. 9, 2013, titled "SPHERICAL MULTICELLULAR AGGREGATES
WITH ENDOGENOUS EXTRACELLULAR MATRIX." Each of these patents and
patent applications are herein incorporated by reference in their
entirety.
INCORPORATION BY REFERENCE
[0003] All publications and patent applications mentioned in this
specification are herein incorporated by reference in their
entirety to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
FIELD
[0004] Described herein are edible (e.g., fit for human
consumption) food products formed from a dehydrated mixture of
cultured cells and a carrier (such as hydrogel), as well as methods
of making and using them to form engineered meat products.
BACKGROUND
[0005] The human body needs protein for growth and maintenance.
Aside from water, protein is the most abundant molecule in the
body. According to U.S. and Canadian Dietary Reference Intake
guidelines, women aged 19-70 need to consume 46 grams of protein
per day, while men aged 19-70 need to consume 56 grams of protein
per day to avoid deficiency. This recommendation, however, is for a
sedentary person free of disease. Protein deficiency can lead to
reduced intelligence or mental retardation as well as contribute to
the prevalence of diseases such as kwashiorkor. Protein deficiency
is a serious problem in developing countries, particularly, in
countries affected by war, famine, and overpopulation. Animal
sources of protein, such as meat, are often a source of the
complete complement of all the essential amino acids in adequate
proportions.
[0006] The nutritional benefits of meat are tempered by potential
associated environmental degradation. According to a 2006 report by
the Food and Agriculture Organization of the United Nations (FAO),
entitled Livestock's Long Shadow--Environmental Issues and Options,
the livestock industry is one of the largest contributors to
environmental degradation worldwide, and modern practices of
raising animals for food contributes widely to air and water
pollution, land degradation, climate change, and loss of
biodiversity. The production and consumption of meat and other
animal sources of protein is also associated with the clearing of
rainforests and species extinction. Accordingly, there is a need
for a solution to demands for alternative to meat produced from
live animals.
[0007] Foods such as chips (e.g., chips, crisps, puffs, crackers,
jerky, etc.) are a favorite snack foods in the United States.
Commercially available chips typically contain high amounts of fat
and sodium, and imply high caloric intake. Excessive consumption
may lead to increased health risks, such as of hypertension. For
example, potato chips contain high caloric values, typically
150-160 calories (90-99 of those from fat) per ounce. Baked potato
chips, advertised as a healthier alternative to conventional, fried
chips, typically contain 120 calories, 18 of those from fat in the
same serving size. High caloric intakes, when combined with a
passive lifestyle, can contribute to obesity, hypertension and
peripheral artery disease. In addition, traditional potato chips
generally contain high levels of sodium, in amounts ranging from 7%
to 8% of the daily recommended value based on a 2,000 calorie diet;
a surprising amount, considering an ounce generally consists of
less than 15 chips. High levels of sodium reportedly contribute to
the emergence of conditions such as hypertension, which can lead to
an increased risk of heart attack. Fried potato chips often contain
high amounts of fat and saturated fat, from 10 to 11 grams of fat,
3 of those from saturated fat (representing form 15% to 17% of the
daily recommended value based on a 2,000 calorie diet) per serving.
The high fat content can pose a serious health risk, as high fat
intake can lead to build-up of plaque in arteries, increasing the
propensity for heart attack and stroke. Similarly, consuming excess
fat on a regular basis may increase the risk of diabetes and
obesity.
[0008] There is a need for a snack food, particularly one that may
resemble the widely popular chips, that is high in protein, fiber
and calcium, and low in fat. Although so-called "meat chips" have
been proposed in the past, such products have proven expensive,
lacking in flavor, and, which potentially high in protein, have
also been high in sodium and fat, preventing these from being a
valid alternative to traditional chips. Also importantly, such
"meat chips" have been fabricated from animals grown and
slaughtered in the same manner as most commercially available
meats. As mentioned above, this is both environmentally
problematic, but may also raise moral and ethical issues for
consumers.
[0009] For example, crisp meat-based snacks that resemble potato
chips or other carbohydrate-based snacks are described, for
example, in U.S. Pat. No. 3,497,363, which suggests a crisp fried
meat snack formed by deep-fat frying a freeze-dried slice of meat.
The freeze-drying is said to be critical to the crisp, chewable
nature of the chip. Freeze-drying can be relatively costly on a
commercial scale and deep-fat frying increases the fat content of
the chips, leading to an expensive, high-fat snack. U.S. Pat. No.
3,512,993 proposes mixing meat or seafood with water and a 50/50
blend of potato and corn starch to form a dough that is cooked
under pressure and sliced. The resultant slices are dried and
deep-fat fried before eating. Frying lends the chips a flaky
texture instead of the "hard, horny texture" of the dried chips.
This product is high in fat (with fat contents of 30-40% suggested)
and starch; this makes the chips less desirable to those
controlling caloric and carbohydrate intake from snack foods.
Others have posited approaches for drying sausage slices to make a
snack food without having to fry the slices. U.S. Pat. No.
6,383,549 and U.S. Patent Application Publications 2003/0113433 and
2004/0039727, for example, suggest such processes. Most of these,
however, are not well adapted to commercial-scale production of an
inexpensive snack food and are limited to home-scale batches or
expensive specialty products.
[0010] Described herein are dehydrated foods that may be formed as
chips that address the shortcomings described above.
SUMMARY OF THE DISCLOSURE
[0011] The present invention relates to food products that may be
formed of cultured cells combined (e.g., mixed) with a hydrogel
(e.g., a plant-derived polysaccharide or polysaccharide-based
hydrogel, such as pectin) that is dehydrated. The food product may
be formed into any traditional dry good food product, including but
not limited to: chips, crackers, bars, cereal, pastas, jerky, etc.
Although the examples described herein illustrate food products and
methods of making food products formed into chips that are similar
to traditional chips, the invention described herein may also be
applied to form other food products. The resulting product may be a
snack food, such as a chip, that is made without harm to an animal,
and is high in protein and low in fat. The food product may provide
a healthy, gluten-free, snack that is rich in protein, fiber and
calcium, and may resemble the texture (e.g., crunchiness and/or
friability) of a traditional chip without being fried or baked,
eliminating the fat, and in particular saturated fat. Also
described herein are methods of making a food product, such as a
chip, from cultured cells (e.g., from animal muscle cells).
[0012] For example, described herein are edible, dehydrated food
products that include cultured animal muscle cells, the cultured
animal muscle cells combined with a plant-derived hydrogel, and a
flavorant, wherein the cultured animal muscle cells and
plant-derived hydrogel are formed as a dehydrated, sheet of
material.
[0013] In general, the cultured cells are mixed in with the
hydrogel, then allowed to set or gel and dehydrated. The mixture
may be homogenous (e.g., relatively uniform) or non-uniform, as
cells may clump. The cells may be non-adherent when mixed, or they
may be clustered, for example, in small clusters or clumps. Thus,
the mixture may include individual cells and/or clusters of cells
distributed through the hydrogel that is then dehydrated. In the
final food product, the cultured animal muscle cells and
plant-derived hydrogel may be distributed throughout the sheet of
material.
[0014] The flavorant may be coated onto the food product and/or
within it (included in the mix with the cultured cells and the
hydrogel). The flavorant may be added before, during or after
dehydration.
[0015] For example, described herein are snack foods such as chips.
An edible snack chip may include: cultured animal muscle cells, the
cultured animal muscle cells combined with a plant-derived
hydrogel; and a flavorant, wherein the cultured animal muscle cells
and plant-derived hydrogel are formed as a dehydrated, sheet of
material.
[0016] Any appropriate cultured animal muscle cells may be used.
For example, cultured muscle cells (myocytes) may be derived from
one or more of: beef, veal, pork, chicken, or fish. The cultured
animal muscle cells may comprise one or more of: skeletal myocytes,
smooth myocytes, and cardiac myocytes (or mixtures thereof). The
cultured cells may be all myocytes or a majority of cultured
myocytes. For example, the cellular component of the food product
may have greater than 70 percent myocytes, greater than 80 percent
myocytes, greater than 85 percent myocytes, greater than 90 percent
myocytes, greater than 95 percent myocytes, greater than 98 percent
myocytes, greater than 99 percent myocytes, etc.
[0017] Any appropriate hydrogel, and particularly a plant-derived
polysaccharide, may be used. For example, the plant-derived
polysaccharide may comprise pectin. The plant-derived
polysaccharide (polysaccharide-based hydrogel) may be a low methyl
(LM) esterified pectin. In general, a plant-derived hydrogel is a
hydrogel that originated from a non-animal source. For example, the
plant-derived hydrogel may have been extracted, purified or
otherwise acquired from a plant source. A plant-derived hydrogel
may also be a hydrogel that was identified as a natural product of
a plant. Although the plant-derived hydrogel may have been
identified from a plant source, the immediate source of a
plant-derived hydrogel used in the food products described herein
may be synthetic, for example, the plant-derived hydrogel may be
synthesized or refined. Any of the plant-derived hydrogels
described herein may also be replaced and/or mixed with a hydrogel
of non-plant origin.
[0018] For example, an edible, dehydrated food product as described
herein may comprise: cultured animal muscle cells, the cultured
animal muscle cells combined with a plant-derived hydrogel; and
(optionally) a flavorant; wherein the cultured animal muscle cells
and plant-derived hydrogel are formed as a dehydrated sheet of
material.
[0019] The cultured animal muscle cells and plant-derived hydrogel
in the food product may be distributed throughout the sheet of
material so that a section through the sheet of material has
discrete muscle cells (cultured muscle cells) that may have a
diameter of between about 2 .mu.m to 50 .mu.m (e.g., 2 .mu.m to 40
.mu.m, 2 .mu.m to 35 .mu.m, 5 .mu.m to 50 .mu.m, 5 .mu.m to 40
.mu.m, 5 .mu.m to 30 .mu.m, etc.) and may be distributed through
the section. The cultured muscle cells may be identified
morphologically, and by their expression of markers for muscle
proteins, which may be seen ultrastructurally. For example, the
cultured animal muscle cells and plant-derived hydrogel may be
distributed throughout the sheet of material so that a section
through the sheet of material has a pattern of dehydrated cultured
muscle cells. The cultured muscle cells may be relatively intact,
even after dehydration, and their origin as a cultured cells may be
confirmed by one or more markers, for example by identifying the
pattern of muscle proteins such as actin and myosin within the
dehydrated cultured muscle cells. Thus, even in the dehydrated food
product, a section through the food product will show a distinctive
pattern resulting from the use of a mixture of cultured cells and
plant-derived
[0020] Any of the food products ("chips") described herein may also
include edible microcarriers onto which the cultured animal muscle
cells are grown.
[0021] As used herein the term dehydration or dehydrated in the
context of the food product may refer to the removal of water from
the food product, and in particular, the removal of a majority of
water from the food product compared to a non-dehydrated form of
the food product, so that the moisture content of the food product
is less than, e.g., 70% (e.g., less than 65%, 75%, 80%, 85%, 90%,
95%, etc.).
[0022] In some variations, an edible, dehydrated food product
configured as a snack chip includes: cultured animal muscle cells,
the cultured animal muscle cells combined with a plant-derived
hydrogel (e.g., polysaccharide); and a flavorant, wherein the
cultured animal muscle cells and plant-derived hydrogel are
arranged in sheet of material that is dehydrated with clusters of
muscle proteins having a diameter of between 2 and 50 .mu.m (e.g.,
2-40 .mu.m, 2-30 .mu.m, 2-20 .mu.m, 5-50 .mu.m, 5-40 .mu.m, 5-30
.mu.m, etc.) distributed through the sheet.
[0023] The edible food products described herein may also be formed
or shaped into an easily consumed form, and may resemble a
traditional snack food (e.g., potato chip, stick, pretzel, etc.).
For example, the thickness (or height) may be generally much
smaller than a surface dimension such as the width and length
(e.g., breadth, diameter, etc.). In some variations the edible body
of the chip may have a diameter (e.g., surface/sheet diameter) that
is more than ten times the thickness.
[0024] Also described herein are methods of forming edible food
products. For example, described herein are methods of forming an
edible food product, the method comprising: combining cultured
muscle cells and plant-derived hydrogel to form a mixture; and
dehydrating the mixture to form an edible sheet of material.
[0025] For example, the methods may be used to form edible snack
foods. For example a method of forming a snack chip may include:
combining cultured muscle cells and plant-derived hydrogel to form
a mixture; and dehydrating the mixture to form a chip.
[0026] Any of these methods may also include adding a flavorant.
The flavorant may be added during the mixing step, or may be mixed
with any of the components (e.g., the cultured cells) before the
mixing step. For example, the step of combining may include adding
a flavorant to the mixture of muscle cells and plant-derived
hydrogel. The flavorant may be added after mixing. For example, the
flavorant may be added before dehydrating, during dehydration or
after dehydration. Flavorant may be coated onto the food
product.
[0027] In general, the step of combining may include combining one
or more of cultured skeletal myocytes, smooth myocytes, and cardiac
myocytes with the plant-derived hydrogel to form the mixture. As
mentioned above, any appropriate cell type may be included.
[0028] In some variations additional components may also be mixed
with the cells and hydrogel. For example, combining may include
combining cultured muscle cells and plant-derived hydrogel, and a
calcium chloride solution. Calcium chloride solution may both help
in gelling the hydrogel, and may also add calcium to the food
product, which may be beneficial. For example, combining may
include combining cultured muscle cells and plant-derived hydrogel,
flavorant and a calcium chloride solution.
[0029] Any of these methods may include the step of harvesting the
cultured cells from a tissue culture chamber and washing the cells
before combining with the plant-derived hydrogel. The cultured
cells may be washed by repeatedly rinsing and spinning (e.g.,
centrifuging) to pellet and remove the wash solution. In some
variations the cells may be taken up and/or rinsed in a solution
including flavorant.
[0030] In any of the methods described herein, the cells,
immediately prior to mixing, may be alive, dead or dying. Thus, the
washing and/or mixing may reduce cell viability without affecting
the quality of the final food product (e.g., taste, texture,
nutritional content). However, in some variations, the cells may be
kept alive until dehydration.
[0031] Any of the methods described herein may also include
spreading the mixture onto a surface (e.g., mold) and allowing the
plant-derived hydrogel to gel before dehydrating. The dehydration
step may be performed on the same surface (e.g., mold) or they may
be transferred to a different surface. Any appropriate mold
(including coated molds) may be used. For example, the mold may be
a flat surface (e.g., foil, polymer, paper, etc.). In some
variations the mold may be adapted for use with the dehydrator. For
example, the mold may be thermally conductive and/or vented or
otherwise moisture-permiable.
[0032] In some variations, a method of forming a snack chip may
include: combining cultured muscle cells, plant-derived hydrogel
and flavorant to form a mixture; and dehydrating the mixture to
form a brittle (e.g. friable) chip.
[0033] For example, a method of forming an edible food product may
include: combining cultured muscle cells and plant-derived hydrogel
to form a mixture; and dehydrating the mixture to form an edible
sheet of material. The plant-derived hydrogel may be configured as
an edible microcarrier (which may also include a polypeptide
including a cell-attachment motif) onto which the cultured muscle
cells are grown. Alternatively or additionally, in some variations,
combining comprises combining cultured muscle cells grown on an
edible microcarrier with the plant-derived hydrogel to form the
mixture.
[0034] Combining may comprise combining cultured muscle cells and
plant-derived hydrogel, and a calcium chloride solution and
allowing the plant-derived hydrogel to set (e.g., gel). Any of
these methods may include spreading (e.g., pouring, coating,
spraying, etc.) the mixture onto a surface and allowing the
plant-derived hydrogel to gel before dehydrating.
[0035] In addition, in any of these methods, dehydrating may
include dehydrating the mixture to form a brittle (e.g., friable)
chip.
[0036] For example, described herein are methods of forming an
edible food product into a snack chip, the method comprising:
combining cultured muscle cells and plant-derived hydrogel to form
a mixture; spreading the mixture onto a surface in a layer;
allowing the mixture to set; and dehydrating the mixture to form a
chip.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The novel features of the invention are set forth with
particularity in the claims that follow. A better understanding of
the features and advantages of the present invention will be
obtained by reference to the following detailed description that
sets forth illustrative embodiments, in which the principles of the
invention are utilized, and the accompanying drawings of which:
[0038] FIGS. 1A-1D illustrate components that may be combined to
form the food products described herein. FIG. 1A shows a pellet of
cultured muscle cells (approximately 500 million cells), shown in
greater detail in FIG. 1B. FIG. 1C shows a flavored vegetable broth
(including flavorant) and FIG. 1D shows a 4% solution of
pectin.
[0039] FIGS. 2A and 2B illustrate the formation of a food product
configured as a chip. In FIG. 2A, the food product has been formed
by combining the components illustrated in FIGS. 1A-1D along with a
calcium chloride solution; this mixture is spread onto a mold,
allowed to gel, then dehydrated. FIG. 2B illustrates the dehydrated
food product resulting.
[0040] FIGS. 3A1 and 3A2 show top and side views, respectively, of
one configuration of a food product, configured as a chip, as
described herein. FIGS. 3B1 and 3B2 show top and side views,
respectively, of another configuration of a food product configured
as a thin oval chip. FIGS. 3C1 and 3C2 show top and side views,
respectively, of another configuration of a food product configured
as a thin triangular chip.
[0041] FIG. 4 shows a transverse section through a dehydrated chip
as described herein. The section has been stained using anti-alpha
smooth muscle actin (SMA) antibodies. The SMA appears as darker,
somewhat circular (cellular) shapes. The histology indicates a
characteristic distribution of the muscle proteins (e.g., actin)
within the cultured cells forming the chip, showing the pattern of
dehydrated cultured muscle cells mixed with the animal-based
hydrogel.
[0042] FIG. 5 is a table showing a compositional analysis of one
example of a dried food product (chip) as described herein, showing
the high percentage (e.g., greater than 50%) of protein. The
composition may also indicate the presence of the animal-based
hydrogel, and shows that in this example the dehydrated food
product has a moisture content of less than 5% (e.g., 4.01% in this
example) for the body of the dehydrated food product.
DETAILED DESCRIPTION
[0043] In general, described herein are food products formed from
cultured cells, and particularly cultured muscle cells (myocytes)
grown in vitro, without requiring further processing of the
originating animal. The cultured cells may be grown using culture
media that does not originate from animal sources (e.g.
plant-derived, yeast-derived, single-cell derived, etc.). Further,
the food products described herein may be formed by combining
cultured cells with a hydrogel to form a mixture, allowing the
mixture to gel, and dehydrating the resulting mixture to form the
edible food product. The shape and/or flavor of the edible food
product may be manipulated to determine the type of food product
formed, including chips, crackers, bars, cereal, pastas, etc. One
or more flavorants and/or fortifying agents may be added before,
during, or after combining the cultured cells and hydrogel and
dehydrating.
[0044] In general, any appropriate method of culturing cells may be
used, including culturing on a surface, solution, bioreactor, etc.
Cultured cells are typically muscle cells, such as non-human
myocytes, though other cell types may be used. Cells may originate
from any appropriate source. For example, suitable cells may be
derived from mammals such as antelope, bear, beaver, bison, boar,
camel, caribou, cattle, deer, elephant, elk, fox, giraffe, goat,
hare, horse, ibex, kangaroo, lion, llama, moose, peccary, pig,
rabbit, seal, sheep, squirrel, tiger, whale, yak, and zebra, or
combinations thereof. In some embodiments, suitable cells are
derived from birds such as chicken, duck, emu, goose, grouse,
ostrich, pheasant, pigeon, quail, and turkey, or combinations
thereof. In some embodiments, suitable cells are derived from
reptiles such as turtle, snake, crocodile, and alligator, or
combinations thereof. In some embodiments, suitable cells are
derived from fish such as anchovy, bass, catfish, carp, cod, eel,
flounder, fugu, grouper, haddock, halibut, herring, mackerel, mahi
mahi, marlin, orange roughy, perch, pike, pollock, salmon, sardine,
shark, snapper, sole, swordfish, tilapia, trout, tuna, and walleye,
or combinations thereof. In some embodiments, suitable cells are
derived from crustaceans such as crab, crayfish, lobster, prawn,
and shrimp, or combinations thereof. In some embodiments, suitable
cells are derived from mollusks such as abalone, clam, conch,
mussel, oyster, scallop, and snail, or combinations thereof. In
some embodiments, suitable cells are derived from cephalopods such
as cuttlefish, octopus, and squid, or combinations thereof. In some
embodiments, suitable cells are derived from insects such as ants,
bees, beetles, butterflies, cockroaches, crickets, damselflies,
dragonflies, earwigs, fleas, flies, grasshoppers, mantids,
mayflies, moths, silverfish, termites, wasps, or combinations
thereof. In some embodiments, suitable cells are derived from
non-arthropod invertebrates (e.g., worms) such as flatworms,
tapeworms, flukes, threadworms, roundworms, hookworms, segmented
worms (e.g., earthworms, bristle worms, etc.), or combinations
thereof. The cultured cells may be native or modified (e.g.,
transgenic).
[0045] In general, the cultured cells may be grown to a sufficient
density, harvested, and washed before combining with other
components of the food product mixture (including the hydrogel).
Washing may remove media, and may be performed in water (including
buffered solutions, such as PBS). Cultured cells may be repeatedly
pelleted (e.g. by centrifugation) and rinsed to wash. In some
variations cells may be cultured with a microcarrier, and in
particular with an edible microcarrier. As will be described in
greater detail below, the edible microcarrier may be an edible
plant-derived polysaccharides which may also include a polypeptide
including a cell-attachment motif. A plant-derived hydrogel may be
used combined with cells grown on edible microcarriers as described
herein for cells grown without edible microcarriers; alternatively
in some variations no additional hydrogel is added and the edible
microcarrier and cells alone may be used to form the chip.
[0046] As mentioned above, any appropriate hydrogel may be used. In
general, the hydrogel must be edible (e.g., safe for human
consumption). The hydrogel may include a polysaccharide that can be
cross-linked, such as a pectin. For example, one class of
polysaccharides that may be used are low methyl (LM) esterified
pectins, an abundant plant derivate already used in food
[0047] Any of the food products described herein may be referred to
as dried food products or dehydrated food products.
[0048] Any of the food products described herein may include one or
more flavorants. The term "flavorant" may mean both natural and
artificial varieties. This is intended to include "natural
flavorants" as defined, for example, by Title 21 of the U.S. Code
of Federal Regulations, namely essential oils, oleoresins, essence
or extractive, protein hydrolysates, distillates, or any product of
roasting, heating or enzymolysis, which contains the flavoring
constituents derived from a spice, fruit or fruit juice, vegetable
or vegetable juice, edible yeast, herb, bark, bud, root, leaf or
any other edible portions of a plant, meat, seafood, poultry, eggs,
dairy products, or fermentation products thereof, whose primary
function in food is flavoring rather than nutritional (21 CFR
101.22).
[0049] Flavorant(s) may also include "artificial flavorants", in
particular, chemically synthesized compounds of natural flavorants
that do not necessarily meet the specifications stated above.
Artificial flavorants may include chemical compounds found in
"natural flavorants."
[0050] In addition, "flavorant" may also be a general term to
denote an agent that imparts taste, flavor aromatics, and feeling
factors. Tastes are sensations that are processed through receptors
on the tongue, and generally include salt, sweet, sour, and bitter.
Flavor aromatics are those flavor volatiles emitted while biting,
chewing, drinking and swallowing food, and are sensed by the
olfactory receptors. Feeling factors, in the language of flavor,
describe sensations perceived in the mouth, on the tongue, or in
the nasal passages (or anywhere in the oral/nasal cavities). These
sensations may be separate and distinct from tastes, salt, sweet,
sour and bitter, and from the myriad of flavor aromatics perceived
by the olfactory sense. Compounds which produce these sensations
vary in volatility but many are susceptible to vapor phase
transfer. Such feeling factors include the pungency of "smoke"
flavors, astringency of fruits, cooling of mints, or the heat of
peppers. More specifically, a flavorant may enhance or change the
taste or the aroma of an item, or both the taste and aroma. This
change may be to either enhance a desired taste or flavor, or mask
an undesirable taste or aroma. It should be appreciated that
flavorants, in most applications, are non-toxic and ingestible.
[0051] Flavorants may include flavor aromatics, although some
components of flavorants do not possess olfactory stimulating
properties. For example, flavoring condiments, some spices and
seasonings, including artificial sweeteners, while lacking
olfactory stimulating properties, are nevertheless useful
flavorants in practicing the present invention. Certain spices or
mixtures of spices for flavoring packaged snack foods, including
such representative examples as potato chips, corn chips, barbecue
chips, cheese crackers, as well as others, may be seasoned with
homogeneous and heterogeneous combinations of solid or particulate
spices and condiments, such as a spicy barbecue flavorant. They
possess flavorant (taste) enhancing properties, and therefore, are
useful flavorants along with other spices commonly applied to
foodstuff as flavoring agents in manufacturing processes.
[0052] The following is not a comprehensive list, but is only
representative of some common taste flavorants, plus some sensation
producing flavorants. Examples of taste and sensation producing
flavorants include, artificial sweeteners, glutamic acid salts,
glycine salts, guanylic acid salts, inosinic acid salts,
ribonucleotide salts, and organic acids, including acetic acid,
citric acid, malic acid, tartaric acid, polyphenolics, and so
on.
[0053] This list is only exemplary of common flavor aromatics.
There are thousands of molecular compounds that may be combined or
used independently to create a particular desired flavor. A few
representative examples of common flavor aromatics include isoamyl
acetate (banana), cinnamic aldehyde (cinnamon), ethyl propionate
(fruity), limonene (orange), ethyl-(E,Z)-2,4-decadienoate (pear),
allyl hexanoate (pineapple), ethyl maltol (sugar, cotton candy),
methyl salicylate (wintergreen), and mixtures thereof.
[0054] Any of the food products described herein may also include
one or more fortifying agents. A fortifying agent may be a vitamin,
mineral, or the like, including any appropriate micronutrients.
Examples may include, but are not limited to essential trace
elements, vitamins, co-vitamins, essential fatty acids, essential
amino acids, photonutrients, enzymes, etc.
[0055] As mentioned above, any of the food products and methods of
forming them may be formed into any appropriate form factor. For
example, the food products may be configured as a chip (e.g., the
form factor of a potato chip, tortilla, or crisp). As used herein,
a chip may refer generally to a thin piece of food (typically
consumed by hand) that is often made crisp by being fried, baked,
or dried and typically eaten as a snack, or as part of a meal.
EXAMPLE
[0056] In one example, a method of forming a dehydrated food
product from cultured cells is adapted to produce chips with high
animal protein content.
[0057] Edible chips are prepared by dehydrating a mixture of animal
cells and hydrogel solution such as a plant-derived polysaccharide.
One example of a polysaccharide that may be used is a low methyl
esterified pectin, an abundant plant derivate already used in food.
For the flavoring of the chips, specially-prepared vegetable broth
and/or seasoning may be added. The mixture may be spread over a
mold (e.g., a parchment paper mold) and dehydrated, e.g., in a food
dehydrator, to enhance flavor and obtain crunchiness.
[0058] FIGS. 1A-1D illustrate components that may be combined to
form the food product. The ingredients entering in the composition
of each of the chips in this example, include approximately 500
million cells (shown pelleted in FIGS. 1A and 1B), 800 microliters
of a flavored broth (FIG. 1C) and 300 microliters of a 4% solution
of plant-derived hydrogel (the polypeptide pectin in this example,
shown in FIG. 1D). The addition of calcium may allow the pectin to
gel after the mixture is spread on a surface such as a parchment
paper mold, as shown in FIG. 2A. After the hydrogel is allowed to
set, it may be dehydrated. Dehydration may be performed at
60.degree. C. for 19 hours. The final product is a crispy, flavored
chip (shown in FIG. 2B).
[0059] The food product (which may be referred to herein as a chip,
edible snack chip, cultured cell snack chip, or the like) may be of
any shape, including the shape of traditional "chips", including
square, rectangular, triangular, oval, circular, or the like, and
may be flat, bent or curved. Shapes may be formed by the
dehydration process (e.g., on the mold). Once dehydrated, the
shapes may be removed from the substrate (molding surface) and
further processed. Further processing may include adding additional
flavoring, including adding salt, sugar, etc. or edible
coatings.
[0060] For example, FIGS. 3A1-3C2 illustrate examples of shapes of
cultured cell snack chips that may be formed as described herein.
Any appropriate size and shape may be formed as described herein in
Example 1. In this example, the shapes are planar (though curved or
bent); more complex shapes may also be formed. For example, FIG.
3A1 shows a cultured cell snack chip formed to have a ridged,
somewhat irregular-appearing shape, similar to a traditional ridged
potato chip. FIG. 3A2 shows a side view of this shape. Similarly,
FIG. 3B1 shows a cultured cell snack chip having an oval shape,
while FIG. 3B2 shows a side view of the chip. FIG. 3C1 shows a chip
having a triangular shape and a relatively thin cross-section (FIG.
3C2). As mentioned, the cultured cell snack chips may be formed of
any size (surface diameter, breadth, length, etc.) and thickness,
including sizes and thicknesses that may be readily eaten by hand.
For example the cultured cell snack chips may have a size (e.g.,
average or in some variations median surface diameter) of between
about 1 cm and 15 cm (e.g., between about 2 cm and 10 cm, etc.) and
a thickness of between about 0.1 mm and 10 mm (e.g., 0.5 mm and 5
mm, 0.5 mm and 4 mm, 0.5 mm and 3 mm, 0.5 mm and 2 mm, etc.).
[0061] Cultured muscle cells may be combined with the plant-derived
hydrogel solution either immediately before dehydrating, or they
may be cultured with a hydrogel. In some variations, as mentioned
above, cells may be cultured on edible microcarriers that may be
formed of a polysaccharide (which may also include a polypeptide
including a cell-attachment motif) and/or other edible material.
The cells may be muscle cells that are from an established cell
line, including immortalized muscle cells, or they may be primary
cultures, or they may be mixtures of these.
[0062] For example, in vitro cultured cells may be harvested to
form the food product. For example, in the sample chip illustrated
above, a culture of 500 million cells yield may be removed from
CellStack culture chambers (i.e. seeded with 30 million bovine
smooth muscle cells and cultured for 5 days). Cells may be washed
by centrifuging to pellet the cells and rinsing with PBS. The PBS
can then be removed. In this example, 800 microliters of a
specially-prepared broth (spicy teriyaki sauce) may be added to the
cells, and 300 microliters of a pectin solution (4% in distilled
water) warmed up at 70.degree. C. may also be added. The cell,
broth and pectin mixture may be mixed (e.g., using an Eppendorf
combitip), by vortexing, etc. Approximately 50 microliters of
calcium chloride solution (0.5M in water) may then be added and the
mixture again mixed (e.g., using an Eppendorf combitip to
homogenize). Some air can be incorporated at this step.
[0063] The mixture may then be distributed into a parchment paper
mold that has been sprayed with 50 microliters of calcium chloride
solution (0.5M in water), similar to that shown in FIG. 2A-2B. The
pectin is allowed to gel for 5 minutes at room temperature.
Thereafter, the mold and gel may be placed in a dehydrator at
60.degree. C. for 19 hours to dehydrate the food product and form
the chip. The chip may then be removed from the mold (parchment
paper).
[0064] This method may be scaled up and/or automated to from
multiple chips. As mentioned, the method may also be modified to
form other food products.
[0065] The methods of forming the cultured cell snack chips
described herein typically results in a chips that is structurally
distinct from existing food products. This is apparent when
examining the ultrastructural properties of the resulting chip. For
example, a section through a cultured cell snack chip shows the
dehydrated cultured muscle cells within a matrix of the dehydrated
hydrogel. The identity of the cultured muscle cells may be
confirmed by staining for identifying markers, including protein
markers (e.g., actin, myosin, etc.). For example, in a section
through the dried edible food product shown in FIG. 4, the
ultrastructure shows residual cellular shapes (e.g., cultured
muscle cells) distributed within the dehydrated hydrogel. In
general, in a dehydrated chip the ultrastructure may show discrete
and dehydrated cultured cells (or clusters of cells) that express
specific markers such as animal protein markers. These cells are
generally mixed with the plant-derived hydrogel. These chips may
also be referred to as animal protein-containing chips.
Visualization may show a distinct pattern of the cultured cells (or
dehydrated remains of the cultured cells) within the plant-derived
hydrogel. The cultured muscle cells may still be apparent (and
reasonably intact) in the dehydrated product.
[0066] In FIG. 4, a section through one example of a dried chip
formed as described herein shows smooth muscle actin (detected
through reaction with alpha smooth muscle actin antibodies). The
pattern of discrete cellular-shaped bodies in the dehydrated sample
is characteristic of the cultured procedure used to form the chip,
i.e., mixing cultured cells with hydrogel and dehydrating the
mixture in a sheet.
[0067] The composition of the edible food products formed as chip
snacks may also be characteristic compared to other edible food
products that are not formed of cultured cells mixed with hydrogel
and dehydrated. For example, the table of FIG. 5 shows results of a
nutritional analysis of a chip formed as described herein, and
shows that it has a high protein content of (in this example,
approximately 70%, though it may more generally be between about
40% and 90%, (e.g., between about 40% and 80%, between about 45%
and 90%, between about 45% and 80% between about 50% and 90%,
between about 50% and 80%, between about 60% and 90%, between about
60% and 80%, greater than 50%, greater than 55%, greater than 60%,
greater than 65%, greater than 70%, greater than 75%, greater than
80%, etc.).
Edible Microcarriers
[0068] As mentioned above, any of the food products described
herein may use cultured cells grown on edible microcarriers
(including microbeads). Edible microcarrier may be formed of an
animal-product-free material or materials, meaning the material or
materials are derived from non-animal (including plant) sources.
The edible microcarrier may typically be formed of a material that
is edible (nutritious and/or safely digestible in large quantities)
and a material having a cell-attachment domain or motif. In some
variations the edible microcarrier may be formed, at least in part,
by a cross-linked structure of polysaccharide and a polypeptide
including a cell-attachment motif (such as RGD). As a specific
example, the edible microcarrier may be formed by cross-linking a
pectin (e.g., thiol-modified pectin, PTP) and an RGD-containing
polypeptide such as the cardosins.
[0069] Any of the edible microcarriers described herein may also
include additional (supplemental) material, including flavorants
(additives for enhancing the flavor), additives for enhancing
appearance and/or nutritive value of the edible microcarrier, and
the resulting food product (e.g., chip) fabricated using the edible
microcarriers. These additives (e.g., flavorants) may be used in
place of, or in addition to, flavorants added prior to or after
dehydrating.
[0070] For example, edible microcarriers may include edible
microsponges and/or edible microbeads. These microcarriers may be
porous (e.g., sponge-like) or smooth. The edible microcarriers for
use in forming engineered meat may be formed into
microbeads/microparticles for use in a bioreactor, and may be
between about 3 mm and about 0.02 mm in diameter (e.g., between
about 2 mm and about 0.05 mm, between about 1 mm and 0.1 mm,
between about 1 mm and 0.3 mm, etc.). For example, the microbeads
may be around 0.5 mm in diameter. The size may represent an average
or median size, or a maximum/minimum size. The shape of the
microcarriers may be regular (e.g., spherical, rounded, etc.) or
irregular, for example spherical, cubic, or the like; any of these
shapes may be porous.
[0071] Edible microcarriers may be made by any appropriate process,
including molding, extrusion, injection, infusion, etc. of the
material forming the edible microcarrier. Edible, highly porous
microcarriers that can be used in cell culture techniques with the
bioreactors and remain integral part of the final engineered
comestible product (e.g., chip) may be formed from edible
animal-free materials (including cultured animal cells that may be
from long-term cultures or may be removed without killing the
animal). Such edible microcarriers may be prepared by forming the
components, e.g., polysaccharide and polypeptide, into a
cross-linked hydrogel, lyophilization of cross-linked hydrogel, and
shaping (e.g., cutting) the lyophilized gel into appropriate
sizes.
[0072] One example of a method of forming an edible microcarrier
includes forming the principle components of the microcarrier, the
polysaccharide and polypeptide. For example, one class of
polysaccharides that may be used are low methyl (LM) esterified
pectins, an abundant plant derivate already used in food. For
example the LM esterified pectin used may be derived to form a
thiol-modified pectin (PTP) that is 100% edible and digestible.
Thiol functions are found in garlic and onion. One class of
polypeptides that may be used includes the cardosins. Cardosins are
aspartic proteinases that may be extracted from Cynara cardunculus
L., and that contain cell binding RGD motifs that promote cell
attachment. For example, cardosins may be derivatized through their
cysteines to introduce new thiol groups. Cardosins are already used
by the food industry, specifically in cheese-making. In other
variations, the cardosins could be substituted (or supplemented) by
another polypeptide, including a synthetic peptide, with an RGD
sequence that is edible.
[0073] In some variations, PTP and derivatized cardosins may be
cross-linked through oxidative disulfide bond formation. In this
example, PTP-cardosin hydrogel may be cross-linked under mild
conditions using (the oxidized form of) glutathione disulfide
(GSSG) obtained by bubbling air into a solution of high-grade
glutathione (GSH, e.g., such as health-food store grade
glutathione). Additional additive (e.g., flavorings, nutrients,
colors, etc.) may be added as well.
[0074] The hydrogel may then be shaped or formed. For example,
macrosponges (1-5 mm thick) may be formed by casting the hydrogel
solution in molds and allow the cross-linking to continue in air
overnight, then lyophilized and cut to desired dimension (larger
sponges for tissue engineering applications, small fragments of 0.5
mm for bioreactor applications).
[0075] For large scale production of microbeads, a coaxial airflow
bead-making device may be used. For example, beads may be composed
of the modified cross-linked pectin and cardosin hydrogel (e.g.,
PTP-cardosin hydrogel). In one variation, a method of forming
microcarriers of PTP and cardosins may be performed by steps that
may include: (1) creating pectin-thiopropionylamide (PTP) by
derivatization of pectin with cystamine at two levels of
modification (e.g., 10%, 25%) followed by reduction; (2)
introduction of new thiol groups by derivatization of cardosin
(e.g. cardosin A); (3) development of GSSG cross-linked hydrogel in
slab format with PTP and thiolated cardosins (the pH,
concentration, and the like may be optimized for forming the
hydrogel; additives, e.g., coloring, nutrients, etc., may also be
included), and the hydrogel may be lyophilized; (4) creating beads
using a bead generator such as the Nisco coaxial airflow bead
generator and lyophilize the GSSG hydrogel sphere to obtain the
microcarriers.
[0076] In use, the microcarriers may be used to culture cells, for
example smooth muscle cells, in large amounts for forming the
edible materials (e.g., chips). As mentioned above, in general,
other cells types may also be used on the microcarriers in addition
to (or instead) of the muscle cells, including satellite cells,
etc.
[0077] For example, microcarriers as described herein may be seeded
with muscle cells (e.g., smooth muscle cells) and cultured. In
particular, the cells and microcarriers may be cultured in a
bioreactor. The resulting cultures may be grown to a desired level
and used directly to form a chip, without the necessity to separate
or otherwise remove the microcarriers. The chip may be formed as
described above, in which (instead of combining just the cells and
the hydrogel), microcarriers onto which cultured cells have grown
are combined with a hydrogel and dehydrated. In some variations,
the microcarriers with cells may be directly applied (e.g., poured,
sprayed, etc.) onto the forming surface such as a mold prior, to
dehydrating without adding hydrogel; for example, the microcarriers
on the surface may be cultured to allow the cells and/or
microcarriers to at least partially fuse. For example, the
microcarriers with cells may be cultured on a surface for some
amount of time (e.g., for 4 hours, 12 hours, 18 hours, 24 hours, 48
hours, 3 days, etc.) or may be immediately dehydrated.
[0078] In some variations the microcarriers with cells (which may
be grown to density, including to confluence on the microcarriers)
may be added together with additives including flavorant and with a
solution of hydrogel (e.g., plant-derived polysaccharide), similar
to what is shown and described in Example 1, above. Thereafter, the
mixture may be dehydrated.
[0079] For example, the edible microcarriers with cultured cells
may be formed into chips after incubation in the bioreactor for an
appropriate time to allow cells to grow and multiply on the
microcarriers (e.g., 12 hours, 24 hrs, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,
etc.), to form cellularized microcarriers. In general a
cellularized microcarrier is a microcarrier (e.g., edible,
animal-product-free) onto which cells (e.g., muscle cells) have
adhered and grown. As mentioned, the cells on the microcarrier may
be grown to confluence, though this is not required. Further, the
cells may fuse on and/or in the surface of the microcarrier. The
cellularized microcarriers may be at least partially fused.
[0080] In these examples, the body of the food product may also
include microcarriers, which may be visualized (e.g., at
magnification).
[0081] For example, in variations using edible microcarriers, a
method of forming the chips described herein may include culturing
a plurality of muscle cells on edible and animal-product-free
microcarriers in suspension to form a plurality of cellularized
microcarriers. The cellularized microcarrier may be mixed with a
plant-derived hydrogel, and in some variations an additive such as
a flavorant. The mixture of cellularized microcarriers and hydrogel
(either with or without additional additives), may then be placed,
poured, sprayed or otherwise applied onto a substrate (e.g., mold
or other surface) appropriate for use in a dehydrator. The mixture
may then be dehydrated as described above. Additional
flavorants/additives (e.g., salt, etc.) may then be added, and the
chips may be further processed and/or packaged.
[0082] Cells may generally be cultured with any of the edible
microcarriers described herein in a suspension, including in a
bioreactor. For example, cells may be seeded into the media along
with the edible microcarriers and allowed to contact, adhere to,
and grow on the appropriate edible microcarrier. For example,
culturing may include culturing a plurality of muscle cells on
edible and animal-product-free microcarriers comprising a hydrogel
of thiol-modified pectin (PTP) and cardosin. In some variations,
culturing comprises culturing a plurality of muscle cells on edible
and animal-product-free microcarriers wherein the
animal-product-free microcarriers comprise a flavoring, a flavor
enhancer, a colorant, a color enhancer, and a nutritional
enhancer.
[0083] In some variations a cellularized microcarrier is covered
(e.g., greater than 50% covered, greater than 60% covered, greater
than 70% covered, greater than 80% covered, greater than 90%
covered, covered to confluency) with the cells. As described in
U.S. Pat. No. 8,703,216, previously incorporated by reference in
its entirety, the cells used may be one or more types, including in
particular muscle cells. Microcarriers covered to the appropriate
degree with cells (e.g., >50%, >60%, >70%, >80%,
>90%, etc. covered) may be referred to as cellularized
microcarriers.
[0084] Although the use of edible microcarriers as described herein
is optional, it may provide some advantages over traditionally
cultured cells. For example, cells used for the edible chips
described herein, which may include, e.g., smooth muscle cells,
satellite cells, fibroblasts, adipocyte progenitor cells, etc., are
typically anchorage-dependent, and require a surface to attach to.
Current cell culture methods may use flasks, tubes and/or plates
(e.g., cellStacks or hyperflasks) to provide a surface onto which
the cells may adhere and grow, which may result in a manual labor
intensive process, and may require enzymes to detach the cells from
the surface and large volume of medium to yield the cells. Most of
the material is single use, thus generating waste; expansion of the
cells is typically achieved by seeding more plates with larger
number of layers as the culture progresses.
[0085] The microcarriers described herein may provide large surface
area/volume for cell attachment, particularly if they are micro- or
macro-porous. The initial step of cell expansion may include mixing
the cells and the microcarriers in a small bioreactor. The cells
attach and multiply on the microcarriers that are maintained in
suspension. When the maximal growth is achieved, the microcarriers
may be collected and can be used for seeding a bioreactor of larger
volume, or may be used directly if sufficient amounts are grown.
The cells don't have to be separated from the microcarriers, since
the microcarriers described herein are edible, eliminating the use
of enzymes and the risk to damage the cells. The process is time
efficient and easy to scale up. Industrial bioreactors can achieved
large volumes (e.g., greater than 1000 L) in less space than
traditional cell culture incubators.
[0086] Terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. For example, as used herein, the singular forms "a",
"an" and "the" are intended to include the plural forms as well,
unless the context clearly indicates otherwise. It will be further
understood that the terms "comprises" and/or "comprising," when
used in this specification, specify the presence of stated
features, steps, operations, elements, and/or components, but do
not preclude the presence or addition of one or more other
features, steps, operations, elements, components, and/or groups
thereof. As used herein, the term "and/or" includes any and all
combinations of one or more of the associated listed items and may
be abbreviated as "/".
[0087] As used herein in the specification and claims, including as
used in the examples and unless otherwise expressly specified, all
numbers may be read as if prefaced by the word "about" or
"approximately," even if the term does not expressly appear. The
phrase "about" or "approximately" may be used when describing
magnitude and/or position to indicate that the value and/or
position described is within a reasonable expected range of values
and/or positions. For example, a numeric value may have a value
that is +/-0.1% of the stated value (or range of values), +/-1% of
the stated value (or range of values), +/-2% of the stated value
(or range of values), +/-5% of the stated value (or range of
values), +/-10% of the stated value (or range of values), etc. Any
numerical range recited herein is intended to include all
sub-ranges subsumed therein.
[0088] Although various illustrative embodiments are described
above, any of a number of changes may be made to various
embodiments without departing from the scope of the invention as
described by the claims. For example, the order in which various
described method steps are performed may often be changed in
alternative embodiments, and in other alternative embodiments one
or more method steps may be skipped altogether. Optional features
of various device and system embodiments may be included in some
embodiments and not in others. Therefore, the foregoing description
is provided primarily for exemplary purposes and should not be
interpreted to limit the scope of the invention as it is set forth
in the claims.
[0089] The examples and illustrations included herein show, by way
of illustration and not of limitation, specific embodiments in
which the subject matter may be practiced. As mentioned, other
embodiments may be utilized and derived there from, such that
structural and logical substitutions and changes may be made
without departing from the scope of this disclosure. Such
embodiments of the inventive subject matter may be referred to
herein individually or collectively by the term "invention" merely
for convenience and without intending to voluntarily limit the
scope of this application to any single invention or inventive
concept, if more than one is, in fact, disclosed. Thus, although
specific embodiments have been illustrated and described herein,
any arrangement calculated to achieve the same purpose may be
substituted for the specific embodiments shown. This disclosure is
intended to cover any and all adaptations or variations of various
embodiments. Combinations of the above embodiments, and other
embodiments not specifically described herein, will be apparent to
those of skill in the art upon reviewing the above description.
* * * * *