Note: Descriptions are shown in the official language in which they were submitted.
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USE OF CHLOROPLASTS FOR OXYGEN PRODUCTION IN CELL CULTURES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of United States
Provisional
Application No. 62/734,314, filed September 21, 2018, which application is
hereby
incorporated by reference in its entirety.
TECHNICAL FIELD
[001] The disclosure relates generally to producing large quantities of oxygen
for cell cultures
and providing human cells with the ability to photosynthesize.
BACKGROUND
[002] Traditionally, there have been different methods used to isolate a wide
variety of
biological materials, including chloroplasts. For example, a mechanical
support system can be
used to allow for adherence of the isolated biological material, depending
upon the type of
material being isolated. After isolation, tradition methods can use the oxygen
production from
chloroplasts for energy production when introduced to metal. However, these
systems and
methods have failed to effectively incorporate isolated chloroplasts within
human cell lines.
There is a need to incorporate isolated chloroplasts within human cell lines
to allow for
improved oxygen production for a possible cell therapy or biotechnology
production plant.
SUMMARY
[003] The present disclosure provides methods for increasing oxygen
concentration of a
cellular media or cells.
[004] In some aspects, the present disclosure provides a method for increasing
oxygen
concentration in a culture media, the method comprising culturing chloroplasts
in a culture
media including one or more mammalian cells, wherein the chloroplasts remain
external to the
mammalian cells in the culture media; and exposing the chloroplasts to light
to cause oxygen
production by the chloroplasts to enrich the culture media with oxygen to
promote the growth
or differentiation of the mammalian cells.
[005] In some aspects, the present disclosure provides a method for increasing
oxygen
concentration, the method comprising coculturing chloroplasts with one or more
mammalian
cells in a media, wherein the mammalian cells under conditions that cause the
one or more
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mammalian cells to take up the chloroplasts from the media; and increasing
oxygen production
in the mammalian cells by exposing the chloroplasts to light.
[006] In some embodiments, the methods can further comprise isolating
chloroplasts from a
source material. In some embodiments, the mammalian cells are human stem
cells. In some
embodiments, the chloroplasts are provided in a concentration of about 8
million chloroplasts
per ml of media and about 12 million chloroplasts per milliliter of the
culture media. In some
embodiments, the chloroplasts are provided in a concentration of about 9
million chloroplasts
per ml of media and about 11 million chloroplasts per milliliter of the
culture media. In some
embodiments, the chloroplasts produce between about 70 and 90% oxygen in the
culture media.
[007] In some embodiments, the chloroplasts produce between about 80 and 90%
oxygen in
the culture media. In some embodiments, the method may further comprise
maintaining
viability of chloroplasts for at least about 72 hours. In some embodiments,
the methods may
further comprise maintaining viability of chloroplasts for between about 72
hours to about 120
hours. In some embodiments, the methods may further comprise isolating
chloroplasts from a
source material.
[008] In some aspects, the present disclosure provides a culture medium
comprising
chloroplasts in a concentration of about 8 million chloroplasts per ml of
media and about 12
million chloroplasts per milliliter of the culture media. In some embodiments,
the chloroplasts
are provided in a concentration of about 9 million chloroplasts per ml of
media and about 11
million chloroplasts per milliliter of the culture media. In some embodiments,
the chloroplasts
produce between about 70 and 90% oxygen in the culture media. In some
embodiments, the
chloroplasts produce between about 80 and 90% oxygen in the culture media. In
some
embodiments, viability of chloroplasts is maintained for at least about 72
hours. In some
embodiments, viability of chloroplasts is maintained for between about 72
hours to about 120
hours. In some embodiments, the culture medium further comprises mammalian
cells, wherein
increasing oxygen production in the mammalian cells by exposing the
chloroplasts to light.
BRIEF DESCRIPTION OF THE DRAWINGS
[009] The present disclosure is further described in the detailed description
which follows, in
reference to the noted plurality of drawings by way of non-limiting examples
of exemplary
embodiments, in which like reference numerals represent similar parts
throughout the several
views of the drawings, and wherein:
[0010] Figures 1A and 1B depict an example process for producing oxygen for
cell cultures,
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in accordance with the present disclosure;
[0011] Figures 2A and 2B depict images of isolated chloroplasts plated in
wells, in accordance
with the present disclosure;
[0012] Figure 3 depicts a graph showing increased oxygen concentrations in
media including
chloroplasts, in accordance with the present disclosure;
[0013] Figure 4A depicts an image of chloroplast cocultures after the addition
of chloroplasts
to the culture, in accordance with the present disclosure; and
[0014] Figures 4C and 4B depict images of chloroplast cocultures during the
culture period, in
accordance with the present disclosure.
[0015] While the above-identified drawings set forth presently disclosed
embodiments, other
embodiments are also contemplated, as noted in the discussion. This disclosure
presents
illustrative embodiments by way of representation and not limitation. Numerous
other
modifications and embodiments can be devised by those skilled in the art which
fall within the
scope and spirit of the principles of the presently disclosed embodiments.
DETAILED DESCRIPTION
[0016] The present disclosure discusses methods and systems for increasing
oxygen
concentration of cells cultures and cells using isolated chloroplasts. In some
embodiments, the
method involves extracellularly increasing oxygen concentration of human cell
media by
culturing isolated chloroplasts with the human cell media. In some
embodiments, the method
involves intracellularly increasing oxygen concentration of human cells via
coculture of
isolated chloroplasts with human cells, facilitating chloroplast uptake and
sequestration within
cells. By sterilely isolating chloroplast from plant materials and culturing
them in human cell
media, the chloroplasts can remain viable for over 48 hours or, in some
embodiments, over 72
hours in culture and still produce significant amounts of oxygen for use by
the human cells.
[0017] In some embodiments, the methods and systems of the present disclosure
can be utilized
to create temporary chloroplast-human cell symbionts that can conduct
photosynthesis via
coculture of isolated chloroplasts with human Mesenchymal Stem Cells (hMSCs).
In some
embodiments, such symbionts can uptake isolated chloroplasts and conduct
photosynthesis in
vitro and/or in vivo. Thereafter, the symbiotic chloroplast and human cells
can be applied to
different applications, such as for example, insertion into red blood cells
for supporting
mammalian cell growth or organism oxygenation. For example, when inserted into
red blood
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cells, chloroplast can produce and carry oxygen in addition to oxygen normally
contained in
the red blood cells (oxygen supplement). In addition to oxygen, chloroplasts
can function to
produce glucose, amino acids and fatty acids. In some embodiments,
chloroplasts can be
delivered by injection into human skin epithelial cells in vivo, providing
nutrients for
temporary nutritional supplementation.
[0018] In accordance with some embodiments of the present disclosure, a method
for
extracellularly increasing oxygen concentration of a cellular media is
provided. The method
includes sterilely isolating chloroplasts from a source material, culturing
the chloroplasts in at
least one well plate with a media, wherein the chloroplasts remain separate
from other cells in
the media, and incubating and exposing the cultured chloroplasts to light in
the at least one
well plate to increase oxygen concentration. The source material can be
spinach leaves or other
plant material.
[0019] In accordance with some embodiments of the present disclosure, a method
for
intracellularly increasing oxygen concentration of human cells media is
provided. The method
includes sterilely isolating chloroplasts from a source material, coculturing
the chloroplasts in
at least one well plate with human cell in a media, wherein the chloroplasts
are taken up the
human cells in the media, and incubating and exposing the cultured
chloroplasts to light in the
at least one well plate to increase oxygen concentration. The source material
can be spinach
leaves or another plant material. The human cells can be Mesenchymal Stem
Cells (hMSCs)
or dermal fibroblasts (hDF).
PROCESSING STEPS
[0020] Figures 1A and 1B depict example processes 100, 110 for implementing
the methods
of the present disclosure. Specifically, Figure 1A depicts a process 100 for
isolating and
culturing chloroplasts in well plates with media to extracellularly increase
oxygen
concentration of human cells media, as shown in FIGS. 2A and 2B. At step 102
chloroplasts
are sterilely isolated from source materials. At step 104 the chloroplasts are
plated in wells with
a media. At step 106 the cultures (including chloroplasts) are incubated and
exposed to light
for a predetermined period of time. The results of process 100 is an increased
dissolved oxygen
concentration for the cells in the media. The resulting media can then be used
for various
medical implementations. In some embodiments, the chloroplasts alone can be
used to
oxygenate media.
[0021] Figure 1B depicts a process 110 for isolating and coculturing
chloroplasts with human
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cells to intracellularly increase oxygen concentration of human cells media.
In some
embodiments, such human cells can be Mesenchymal Stem Cells (hMSCs), human
dermal
fibroblasts (hDF), etc. In some embodiments, this can facilitate chloroplast
uptake and
sequestration within cells to increase oxygen concentration of human cells
media. At step 112
chloroplasts are sterilely isolated from source martials and are aliquoted
into well plates
containing a medium. In some embodiments, the cells can be induced to take up
chloroplasts
in such a manner that the chloroplasts maintain its functions within a
mammalian cell
cytoplasm.
[0022] At step 114 the chloroplasts are co-cultured with cells (e.g., human
cells) and are
sequestered for a predetermined culture period well plates. The result of
process 110 is the
production of chloroplast-human symbionts for increased oxygen production.
There are many
benefits to this final product. For example, certain therapies require an
oxygen source along
with paracrine signaling from hMSCs, through creation of these symbiotes we
should be able
to achieve this in an acute non-toxic manner. Furthermore, this process could
be applied to
biotechnological production where oxygen levels are highly regulated. In some
embodiments,
chloroplasts can be co-cultured with mammalian cells, but not taken up by the
mammalian
cells.
SOURCE MATERIALS
[0023] The present methods can utilize different source materials for
isolating chloroplast. In
some embodiments, the chloroplast may be isolated from any combination of
plants and algal.
A variety of plants and algal, such as spinach, basil, arabidopsis, and
tobacco, produce
chloroplast and thus can be employed in the present disclosure. In some
embodiments, the
chloroplasts are isolated from spinach leaves.
CHLOROPLAST ISOLATION AND CULTURE
[0024] The isolated chloroplasts can be obtained from the plants or algal
using any
combination of methods. In some embodiments, the chloroplast can be isolated
from the source
material by deveining and finely chopping the source material with a fine
blade (e.g., step 102
of process 100). The chopped source material can be placed into a mortar along
with a grinding
solution and ground with a pestle until a paste-like consistency. For example,
the source
material can be placed in 15 mL of grinding solution (0.33 M Sorbitol, 10 mM
Sodium
Pyrophosphate, 4 mM Magnesium Chloride, 2 mM Ascorbic Acid, pH was adjusted to
6.5).
Thereafter, the paste can be filtered. For example, the paste can be filtered
through two layers
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of sterile cheesecloth and then spun down in a centrifuge at 300g for 1 minute
at 4 C.
Additionally, supernatant resulting from this process can be collected for
further processing.
For example, the supernatant can be collected and subsequently re-spun down in
a centrifuge
at 1000g for 7 minutes at 4 C. The supernatant after the second centrifuge
cycle can be
removed and a pellet containing the chloroplasts can resuspended in a
suspension solution. In
some embodiments, a pellet having chloroplasts can be created by centrifuging
the solution
with the chloroplasts. The pellet containing the chloroplasts can be, for
example, suspended in
mL of suspension solution (0.33 M Sorbitol, 2 mM Ethylenediaminetetraacetic
acid, 1 mM
Magnesium Chloride, 50 mM HEPES, with the pH adjusted to 7.6). After
processing, the
isolated chloroplasts can be counted using a hemocytometer before being used
in subsequent
steps.
[0025] In some embodiments, sterile chloroplasts can be isolated from spinach
leaves and are
plated in a well, with a media therein, for a predetermined period of time for
culturing (e.g.,
step 104 of process 100). For example, spinach cells can be plated in a well
with a
Mesenchymal Stem Cell Growth Medium (MSCGM) for three days, as discussed with
respect
to Figure 1A. Any cell culture medium can be used. In some embodiments, the
medium may
be buffered with HEPES, but not sodium bicarbonate, and will not include
penicillin-
streptomycin. Additionally, Gentamycin, but can be used as an antibiotic and
Amphotericin B
can be used as an anti-fungal. In some embodiments, normal mammalian cell
culture medium
including serums such as Fetal Bovine Serum can be used for these processes.
In some
embodiments, the chloroplasts can be cultured in MSCGM alone or with
additional
chloroplasts. Thereafter, the chloroplasts can be incubated and exposed to
light (e.g., step 106
of process 100).
[0026] Once isolated, the chloroplasts can be used to prepare a culture
medium. For example,
the present disclosure provides a culture medium comprising chloroplasts in a
concentration of
about 8 million chloroplasts per ml of media and about 12 million chloroplasts
per milliliter of
the culture media.
[0027] Figure 2A depicts an image 200 taken at 20X enhancement showing freshly
isolated
chloroplasts 202, from a spinach leaf, plated in wells of a medium 204 at a
concentration of 10
million chloroplasts 202 per mL of MSCGM medium 204. Similarly, Figure 2B
depicts an
image 200 taken at 20X enhancement showing freshly isolated chloroplasts 202,
from a spinach
leaf, plated in wells of a medium 204 at a concentration of 1 million
chloroplasts 202 per mL
of MSCGM medium 204. Within the images 200, 210, the isolated chloroplasts 202
remained
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vibrant green and photosynthetically viable after isolation. In some
embodiments, the
chloroplasts remain viable for at least 72 hours. In some embodiments, the
chloroplasts remain
viable between 48 hours and 120 hours. In some embodiments, the chloroplasts
can remain
viable for between about 72 hours to about 120 hours. In some embodiments, the
isolated
chloroplasts 202 can be cultured in the plated well of medium 204 for a
predetermined period
of time to produce oxygen.
[0028] Figure 3 depicts a chart 300 showing how chloroplasts isolated from
spinach leaf are
able to increase oxygen concentration in media. In some embodiments,
Mesenchymal stem
cell media or DMEM with FBS can be used. In particular, Figure 3 depicts a bar
graph
showing a first bar shows the percentage of oxygen produced with media 204
alone (about
18%) and the second bar shows the percent of oxygen produced with the isolated
chloroplast
202 (about 10 million chloroplasts) in the media 204 (about 82%). The 82%
oxygen level can
be produced from isolated chloroplast within two days after isolation.
[0029] In some embodiments, the media may be supplemented between about 6
million
chloroplasts per ml of media and about 12 million chloroplasts per ml of
media. In some
embodiments, the media may be supplemented between about 8 million
chloroplasts per ml of
media and about 12 million chloroplasts per ml of media. In some embodiments,
the media
may be supplemented between about 9 million chloroplasts per ml of media and
about 11
million chloroplasts per ml of media. In some embodiments, the chloroplasts
can produce
between about 70 and 90% oxygen. In some embodiments, the chloroplasts can
produce
between about 75% and 85% oxygen. In some embodiments, the chloroplasts can
produce
between about 80% and 90% oxygen. The chart in Figure 3 shows that the
presence of
chloroplasts in culture media leads to strongly significant increases in media
dissolved oxygen
levels over the isolated chloroplasts cultured alone in human cell media. This
increase in
oxygen increases the possible applications for the media, as discussed in
greater detail herein.
CHLOROPLAST SYMBIONTS
[0030] In some embodiments, spinach leaves can be utilized to create temporary
chloroplast-
human cell symbionts 206 that can conduct photosynthesis via coculture of
isolated
chloroplasts 202 with human Mesenchymal Stem Cells (hMSCs) 208, or any cell
type that
actively digests small particles so that the cells can uptake isolated
chloroplasts 202, which can
then conduct photosynthesis in vitro.
[0031] To create chloroplast-human symbionts 206, a solution containing
sterilely isolated
chloroplasts 202 can be aliquoted into well plates containing hMSCs. There are
no specific
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steps or processes used for plating the chloroplasts in the wells, the hMSCs
208 and hDFs will
inherently uptake the chloroplasts 202 when cocultured together. For example,
within 30
minutes to 24 hours from the addition of chloroplasts to hMSC culture wells,
chloroplasts will
appear to be "naturally" and endosymbiotically incorporated into the
cytoplasms of the hMSCs,
remaining intracellularly sequestered until at least day 8 of the culture
period. The relationship
between chloroplasts and cells can be 4-7 chloroplasts per cell. After
culturing, in some
embodiments, the symbiotes can be exposed to light through modification of the
incubators
where light strips (e.g., light emitting diodes (LEDs)) were attached to the
rack directly above
the cultures and were turned on for 12-hour periods of time. Chloroplast-human
cell symbiotes
survive up to a week after cocultures begin.
[0032] Figures 4A-4C depict images 400, 410, 420 demonstrating that viable,
photosynthetically-functional chloroplasts 202 are incorporated and
sequestered within hMSCs
(in some capacity) using the process 110 discussed with respect to Figure 1B.
Figure 4A depicts
an image 400, taken at 20X enhancement, of chloroplast-hMSC cocultures 206 24
hours after
the addition of chloroplasts 202 to the culture 204. As shown in Figure 4A,
chloroplasts 202
appear to be incorporated by hMSCs and residing within their cytoplasms.
Similarly, Figure
4B depicts an image 410, taken at 20X enhancement, of chloroplast-hMSC
cocultures 206 on
day 4 of the culture period. As shown in Figure 4B, chloroplasts 202 have
remained sequestered
within hMSCs remain bright green and viable. Some hMSCs have morphologies
resembling
those of hMSC-derived chondrocytes. Figure 4C depicts an image 420, taken at
20X
enhancement, of chloroplast-hMSC cocultures 206 on day 8 of the culture
period. As shown in
Figure 4C, hMSCs still appear to have sequestered chloroplasts 202 within
their cytoplasms,
and hMSCs continue to adopt a spherical morphology characteristic of
chondrocytes. Thus,
chloroplasts are able to be incorporated by hMSCs in vitro and remain
sequestered and
photosynthetically functional for at least 8 days after their uptake.
APPLICATIONS
[0033] There are a myriad of applications of photosynthetic human cells and
efficient, cheap,
and on-demand oxygen production by isolated chloroplasts is only expanding and
currently
unrecognized by the scientific community. Specifically, a lack of oxygen is a
common problem
that results in irreversible tissue death, and by having an isolated
chloroplast population that
could be delivered to such areas would open up a way to acutely treat such
disorders.
Furthermore, the combination of chloroplasts and hMSCs could act as a
combinatory therapy
where chloroplasts could provide oxygen and the hMSCs could provide paracrine
signalling
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which has been shown to have a positive regenerative affect. Chloroplasts also
are non-toxic
to mammalian cells and thusly could be incorporated into biomanufacturing of
pharmaceuticals
where oxygen level regulations are necessary to provide for positive
manufacturing of the drug
products. Additionally, the lack of money, time, and training with which
oxygen concentration
can be increased and photosynthesis can be induced using the chloroplast
protocols discussed
herein.
[0034] Specifically, there are several opportunities for both
photosynthesizing chloroplast-
human cell symbionts and isolated chloroplasts in culture. Specifically,
isolated chloroplasts
could be used to enhance cell cultures with oxygen production, to grow full
organs or tissues
in vitro, to vascularize and regenerate ischemic tissues, to quicken wound
healing, to enhance
human performance and physical ability, to produce oxygen in hypoxic
environments (e.g.
polluted environments or outer space), and to grow clean meat in vitro for
consumption.
Chloroplast-human cell symbionts and the process of endosymbiotic chloroplast
uptake could
be used to facilitate the destruction of cancerous tumours, to deliver
biomolecules (e.g. insulin)
or drugs via genetic engineering of chloroplast DNA and injection GM
chloroplasts into human
tissue, to quickly and easily bring about the differentiation of human stem
cells to a certain
fate, to function as an alternative to dangerous gene therapies, and to create
permanent
chloroplast-human chimeras that are healthier than normal humans due to
adoption of a
glycolytic metabolism. In some embodiments, the chloroplasts can be used for
internal or
external wound healing. In some embodiments, the chloroplasts may be delivered
as part of a
cream, ointment, or lotion that can be applied to an external wound. In some
embodiments, a
wound dressing impregnated with chloroplasts is provided. In some embodiments,
the
chloroplasts may be delivered to an internal wound using, for example, a
biocompatible
polymer.
[0035] The devices and methods of the present disclosure are described in the
following
Examples, which are set forth to aid in the understanding of the disclosure,
and should not be
construed to limit in any way the scope of the disclosure as defined in the
claims which follow
thereafter. The following examples are put forth so as to provide those of
ordinary skill in the
art with a complete disclosure and description of how to make and use the
embodiments of the
present disclosure, and are not intended to limit the scope of what the
inventors regard as their
invention nor are they intended to represent that the experiments below are
all or the only
experiments performed. Efforts have been made to ensure accuracy with respect
to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors and
deviations should be
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accounted for.
EXAMPLES
[0036] Chloroplast Culture for Increased Media Oxygenation - Dissolved Percent
Oxygen
Data for Cultures
[0037] Chloroplasts were sterilely isolated from spinach leaves and plated in
wells with zero,
1 million, 10 million, or 68.5 million chloroplasts per mL of Mesenchymal Stem
Cell Growth
Media (MSCGM) (Lonza, Walkersville, MD). In order to determine if chloroplasts
could
produce measurable amounts of oxygen after 3 days of dark incubation and 16
hours of light
exposure, cultures' dissolved percent oxygen concentrations were measured with
an SDR
Oxygen Sensor (PreSens). Experimental cultures with 68.5 million chloroplasts
in suspension
solution (without antibiotics) had a dissolved oxygen concentration of 39.63%
3.02% in
comparison to the 18.60 % 0.12% dissolved oxygen of the media-alone control
group (p =
0.001). Even more significant, experimental cultures with 10 million
chloroplasts in MSCGM
had 81.82% 2.50% dissolved oxygen, a value significantly greater than that
of both the 68.5
million chloroplast control (p = 1.9 x 10-5) and the media-alone control (p =
1.0 x 10-6). There
was no significant difference between the dissolved oxygen concentrations of
the experimental
cultures with 1 million chloroplasts per mL of MSCGM and the control, MSCGM-
alone
cultures (p = 0.587). Therefore, from this experiment, it can be concluded
that chloroplasts
remain photosynthetically functional, viable, and sterile after being isolated
and cultured for 3
days in human cell media, being able to produce large quantities of oxygen in
vitro for a wide
variety of applications.
[0038] All patents, patent applications, and published references cited herein
are hereby
incorporated by reference in their entirety. It should be emphasized that the
above-described
embodiments of the present disclosure are merely possible examples of
implementations,
merely set forth for a clear understanding of the principles of the
disclosure. Many variations
and modifications may be made to the above-described embodiment(s) without
departing
substantially from the spirit and principles of the disclosure. It can be
appreciated that several
of the above-disclosed and other features and functions, or alternatives
thereof, may be
desirably combined into many other different systems or applications. All such
modifications
and variations are intended to be included herein within the scope of this
disclosure, as fall
within the scope of the appended claims.
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