WO2024025825A1

WO2024025825A1 - Bioreactor systems and methods for electrically stimulating cells

Info

Publication number: WO2024025825A1
Application number: PCT/US2023/028461
Authority: WO
Inventors: Hai-Long Wang; Gregory A. Worrell; Yihua Wang
Original assignee: Mayo Foundation For Medical Education And Research
Priority date: 2022-07-25
Filing date: 2023-07-24
Publication date: 2024-02-01

Abstract

The disclosure relates to bioreactor systems and related methods for electrically stimulating cells. For example, this disclosure relates to bioreactors that include an enclosure configured to block externally originating electromagnetic radiation and a conductor within the interior of the enclosure that is configured to emit an electromagnetic field that stimulates cells.

Description

BIOREACTOR SYSTEMS AND METHODS FOR ELECTRICALLY STIMULATING CELLS

Cross Reference to Related Applications

This application claims the benefit of U.S. Provisional Application Serial No. 63/391,982 filed on July 25, 2022, which is incorporated herein by reference in its entirety.

Field

Background

Electrical stimulation can be used to increase or decrease an amount of one or more cargo molecules present in or on the surface of extracellular vesicles derived from a target cell.

Electrical stimulation may be used to increase or decrease the amount and/or size of extracellular vesicles derived from a target cell.

Summary

The disclosure describes bioreactor systems and related methods for electrically stimulating cells. In some embodiments, the bioreactors include an enclosure defining an interior and configured to block at least some externally originating electromagnetic radiation from reaching the interior; a conductor disposed within the interior of the enclosure and configured to emit an electromagnetic field; a hollow fiber cartridge disposed within the interior of the enclosure containing a housing defining an internal space, a plurality of hollow fibers within the internal space, each of the hollow fibers defining a lumen, a first inlet port and a first outlet port that are each in fluid communication with the lumens of the plurality of hollow fibers, and a second inlet port and a second outlet port that are each in fluid communication with the internal space of the housing that is external of the plurality of hollow fibers; a reservoir configured to contain a fluid; a pump configured to propel the fluid from the reservoir through the lumens of the plurality of hollow fibers via the first inlet port and the first outlet port; and a signal generator configured to deliver pulses of electrical energy to the conductor.

Particular embodiments of the subject matter described in this document can be implemented to realize one or more of the following advantages. For example, the bioreactor system can enable the production of vesicles with controllable vesicle cargo quantity and/or vesicle size by delivering electrical stimulation of cells grown in the bioreactor. The amount of vesicles produced may also be controlled by the electrical stimulation of the cells. The bioreactor system enables a relatively uniform electrical field to be applied to cells grown within the bioreactor system. The bioreactor system can reduce (e.g., prevent) the contamination of cells and/or extracellular vesicles produced by the cells compared to other systems and methods for electrically stimulating cells. The bioreactor system can accommodate a large volume of cultured cells enabling the growth of a large number of cells under electrical stimulation and the generation of a large amount of extracellular vesicles with controllable cargo quantity and/or vesicle size. The bioreactor system can be used with a heating apparatus (e.g., an incubator) to enable the growth of cells at a desired temperature (e.g., 37 °C). The electrical stimulation parameters, including stimulation frequency, field strength, and waveform morphology, can be readily adjusted as desired by a clinician user.

In a first aspect, the disclosure provides a bioreactor system including: an enclosure defining an interior, the enclosure configured to block at least some externally originating electromagnetic radiation from reaching the interior; a conductor disposed within the interior of the enclosure and configured to emit an electromagnetic field; a hollow fiber cartridge disposed within the interior of the enclosure and including: a housing defining an internal space; a plurality of hollow fibers within the internal space, each of the hollow fibers defining a lumen; a first inlet port and a first outlet port that are each in fluid communication with the lumens of the plurality of hollow fibers; and a second inlet port and a second outlet port that are each in fluid communication with the internal space of the housing that is external of the plurality of hollow fibers; a reservoir configured to contain a fluid; a pump configured to propel the fluid from the reservoir through the lumens of the plurality of hollow fibers via the first inlet port and the first outlet port; and a signal generator configured to deliver pulses of electrical energy to the conductor. In some embodiments, the bioreactor system further includes a dielectric material wherein the dielectric material is disposed within the interior of the enclosure and surrounds at least a portion of the hollow fiber cartridge.

In some embodiments, the enclosure is connected to a ground of the signal generator.

In some embodiments, the bioreactor system further includes tubing, wherein the tubing permits fluid communication between the first inlet port and first outlet port of the hollow fiber cartridge and the reservoir.

In some embodiments, the tubing connects: (i) the reservoir and the pump and (ii) the pump and the first inlet port of the hollow fiber cartridge.

In some embodiments, the tubing connects the first outlet port of the hollow fiber cartridge and the reservoir.

In some embodiments, the conductor is mounted to an interior surface of the enclosure, and the conductor and the enclosure are electrically isolated from each other.

In some embodiments, the enclosure defines ports, wherein the ports enable fluid communication of at least one member selected from the group consisting of the first inlet port, the first outlet port, the second inlet port and the second outlet port with a component external to the enclosure.

In some embodiments, the component external to the enclosure includes at least one member selected from the group consisting of the reservoir, the pump, a tube, and a syringe.

In some embodiments, the dielectric material includes a polymer.

In some embodiments, the dielectric material includes polyoxymethylene or Delrin®.

In some embodiments, the enclosure includes copper.

In some embodiments, the conductor includes copper.

In a second aspect, the disclosure provides a method of increasing or decreasing an amount of one or more cargo molecules present in or on a surface of an extracellular vesicle derived from a target cell using the bioreactor system. The method includes electrically stimulating the target cell, wherein the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers, and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced without electrical stimulation.

In a third aspect, the disclosure provides a method of increasing or decreasing an amount of extracellular vesicles derived from a target cell using the bioreactor system. The method includes electrically stimulating the target cell, wherein the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers, and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced without electrical stimulation.

In a fourth aspect, the disclosure provides a method of increasing or decreasing a size of extracellular vesicles derived from a target cell using the bioreactor system The method includes electrically stimulating the target cell, wherein the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers; and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced without electrical stimulation.

In certain embodiments, the electrical stimulation includes an electric field of from 1 mV/mm to 10 mV/mm in the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers.

In certain embodiments, the electrical stimulation includes an electric field of from 4 mV/mm to 6 mV/mm in the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers.

In certain embodiments, in order to cause the electrically stimulation of the target cell, the signal generator generates pulses with a frequency of from 2 Hz to 200 Hz.

In certain embodiments, pulses are generated for a period of from 0.6 seconds to 60 seconds followed by a gap without pulses of from 0.6 seconds to 120 seconds.

In certain embodiments, the pulse period and the gap are alternated repeatedly.

In a fifth aspect, the disclosure provides a method of increasing or decreasing an amount of one or more cargo molecules present in or on a surface of an extracellular vesicle derived from a target cell using the bioreactor system. The method includes disposing the target cell within the enclosure, wherein the target cell is further disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers, and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced in the absence of the enclosure.

In a sixth aspect, the disclosure provides a method of increasing or decreasing an amount of extracellular vesicles derived from a target cell using the bioreactor system. The method includes, disposing the target cell within the enclosure, wherein the target cell is further disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers, and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced in the absence of the enclosure.

In a seventh aspect, the disclosure provides a method of increasing or decreasing a size of extracellular vesicles derived from a target cell using the bioreactor system. The method includes disposing the target cell within the enclosure, wherein the target cell is further disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers, and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced in the absence of the enclosure.

In certain embodiments, the extracellular vesicle derived includes one or more cargo molecules present in or on a surface of the extracellular vesicles.

In certain embodiments, the cargo molecule is a protein.

In certain embodiments, the cargo molecule is a nucleic acid.

In certain embodiments, the nucleic acid is a miRNA.

In certain embodiments, the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers by injection through the second inlet port. In certain embodiments, the extracellular vesicles are removed from within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers through the second outlet port.

In certain embodiments, the fluid is a cell growth medium.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description herein. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Brief Description of the Figures

Figure la schematically depicts an example system that includes an embodiment of the bioreactor.

Figure lb schematically depicts an exploded view of a portion of the bioreactor of Figure la.

Figure 2 schematically depicts a cross-section view of a CAD model of the bioreactor of Figure la.

Figures 3a-c depict electric field modeling results of the system of Figure la.

Figure 4 schematically depicts a protocol or method for electrically stimulating cells and collecting extracellular vesicles using the system of Figure la.

Figure 5 depicts different pulse sequences that can stimulate cells using the system of Figure la.

Figure 6 is a graph of experimental data of UV absorption measurements of extracellular vesicles. Figures 7a-7e graphically illustrate experimental data of nanoflow cytometry measurements of extracellular vesicles.

Figure 8 shows a heat map showing changes in protein expression.

Figures 9a-9d shows graphs of neural networks representing data from four different experimental conditions.

Figures lOa-lOd show scatter plots of distributions of EVs detected in flow cytometry.

Figure 11 shows a bar graph of changes in the distributions of EVs detected in flow cytometry.

Like reference numbers represent corresponding parts throughout.

Detailed Description

Figures la and lb schematically depict an example embodiment of a bioreactor system 1000. The bioreactor system 1000 includes an enclosure 1100 that includes a top 1102 and a bottom 1104. The enclosure 1100 defines an interior that houses a conductor 1700 and a hollow fiber cartridge 1200. The bioreactor system 1000 also includes tubing 1300, a fluid reservoir 1400, a pump 1500, and a cable 1600 that is connected to a signal generator (not shown).

The hollow fiber cartridge 1200 defines an internal space and includes a plurality of hollow fibers in the internal space. Each of the hollow fibers defines a lumen. The hollow fiber cartridge 1200 further includes a first inlet port 1202 and a first outlet port 1204. The ports 1202 and 1204 are in fluid communication with the lumens of the hollow fibers. The hollow fiber cartridge 1200 also includes a second inlet port 1206 and a second outlet port 1208. The ports 1206 and 1208 are in fluid communication with the internal space of the hollow fiber cartridge 1200 (externally to the hollow fibers).

The first inlet port 1202 and first outlet port 1204 of the hollow fiber cartridge 1200 are connected via the tubing 1300 to the fluid reservoir 1400 and the pump 1500. The pump 1500 can circulate fluid from the fluid reservoir 1400 through the tubing 1300 into the first inlet port 1202 of the hollow fiber cartridge 1200, through the lumens of the hollow fibers, out of the first outlet port 1204 of the hollow fiber cartridge 1200, and then through the tubing 1300 back into the fluid reservoir 1400.

As shown in Figure lb, a portion 1010 of the bioreactor system 1000 includes the top

1102 and the bottom 1104 of the enclosure 1100, which can enclose the hollow fiber cartridge 1200. The first inlet port 1202, the first outlet port 1204, the second inlet port 1206 and the second outlet port 1208 may protrude from the enclosure 1100 through openings defined by the enclosure 1100.

The enclosure 1100 is configured to block at least some externally originating electromagnetic radiation from reaching the interior of the enclosure 1100. In that sense, the enclosure 1100 acts as a type of Faraday cage or Faraday shield. In some embodiments, the top 1102 and the bottom 1104 of the enclosure 1100 are made of metal such as, but not limited to, copper. The enclosure 1100 can be electrically grounded (e.g., connected to the ground of the signal generator in some embodiments).

The conductor 1700 is also disposed in the enclosure 1100, but is not in electrical communication with the enclosure 1100. That is, the conductor 1700 is electrically isolated from the enclosure 1100. In some embodiments, the conductor 1700 is a metal plate (e g., a copper plate). The conductor 1700 is electrically connected to the output of the signal generator. Accordingly, the conductor 1700 can be electrically energized by energy output from the signal generator (with the enclosure 1100 acting as the ground). When the conductor 1700 is electrically energized, an electromagnetic field is emitted from the conductor 1700. The hollow fiber cartridge 1200 is positioned within the electromagnetic field emitted from the conductor 1700.

The bioreactor system 1000 also includes a dielectric member made up of a bottom portion 1802 and a top portion 1804. The dielectric member 1802/1804 surrounds at least a portion of the hollow fiber cartridge 1200 to secure the hollow fiber cartridge 1200 in the interior of the enclosure 1100. In some embodiments, the dielectric member 1802/1804 is constructed of a polymer. In some embodiments, the dielectric member 1802/1804 is made of polyoxymethylene. In some embodiments, the dielectric member 1802/1804 is made of Delrin®.

In some embodiments, the fluid contained in the reservoir 1400 and circulated by the pump 1500 through the lumens of the hollow fibers in the hollow fiber cartridge 1200 is a cell growth media. Accordingly, the bioreactor system 1000 can enable nutrients of the cell growth media to flow or osmotically pass through the porous walls of the hollow fibers and to reach, thereby, the cells residing in the internal space of the housing that is external of the plurality of hollow fibers. In this manner, the cells can receive nutrients from the fluid. The bioreactor system 1000 can be used to electrically stimulate the cells to increase or decrease at least one parameter selected from the group consisting of a quantity of a cargo in or on a surface of extracellular vesicles produced by the cells, a quantity of extracellular vesicles produced by the cells and a size of extracellular vesicles produced by the cells, relative to the absence of the electrical stimulation. In certain embodiments, the extracellular vesicles are exosomes. In certain embodiments, the cargo is contained on a surface of the extracellular vesicle. In certain embodiments, the cargo is contained inside the extracellular vesicle. In certain embodiments, the cargo is a protein. In certain embodiments, the cargo is a transmembrane protein. In certain embodiments, the cargo is a lipid. In certain embodiments, the cargo is a nucleic acid. In certain embodiments, the cargo is an RNA. In certain embodiments, the cargo is a miRNA. In certain embodiments, the cargo is an mRNA. In certain embodiments, the cargo is a non-coding RNA.

U.S. Patent Application No. 16/771,323, which is directed to methods for modulating the release and content of extra-cellular vesicles from target cells using electrical stimulation (e.g., using the bioreactor system 1000 described herein), is hereby incorporated by reference in its entirety and for all purposes.

In some embodiments, the target cell can be any cell type. In some embodiments, the target cell can be selected from the group consisting of a mammalian cell, a protozoal cell, an algal cell, a plant cell, a fungal cell, an invertebrate cell, a fish cell, an amphibian cell, a reptile cell, or a bird cell. In some embodiments, the target cell can be a cancer cell. In some embodiments, the target cell can be a human cell. In some embodiments, the target cell can be a mouse cell.

In certain embodiments, the enclosure 1100 may shield the cells being cultured in the bioreactor system 1000 (within hollow fiber cartridge 1200 in the interior of the enclosure 1100) from a source of electromagnetic radiation external to the enclosure 1100. Accordingly, the electromagnetic radiation to which the cells are exposed can be carefully controlled (and exclusively limited to the electromagnetic radiation emitted/created by the conductor 1700).

In certain embodiments, the functionalities of the enclosure 1100 and the conductor 1700 may be used to increase or decrease at least one parameter selected from the group consisting of a quantity of a cargo in or on a surface of extracellular vesicles produced by the cells, a quantity of extracellular vesicles produced by the cells and a size of extracellular vesicles produced by the cells, relative to the absence of the enclosure 1100 and the conductor 1700.

The function generator and conductor 1700 can be energized to generate a substantially uniform electric field within the hollow fiber cartridge 1200. In some embodiments, the energy used to generate the uniform electric fields can have a controllable frequency, pulse width and/or waveform (e.g., sinusoidal, square wave, white noise).

In some embodiments, the function generator generates a sequence of pulses (e.g., refer to Figure 5) that are delivered to the conductor 1700 to create an electromagnetic field to which the cells are exposed. In some embodiments, the pulses are square-wave pulses. In some embodiments, the pulses have a voltage of at least 1 (e.g., at least 3.4, at least 4) volts (V) and at most 10 (e.g., at most 6.8, at most 5) V. In some embodiments, the pulses have a duration of at least 100 (e.g., at least 200, at least 300, at least 500) microseconds (ps) and at most 1000 (e.g., at most 900, at most 800, at most 500) ps. In some embodiments, the frequency of the pulses is at least 2 (e.g., at least 20) hertz (Hz) and at most 200 (e.g., at most 20) Hz. In some embodiments, a gap without electrical stimulation is applied alternatively with a series of pulses. In some embodiments, the gap has the same duration as the series of pulses. In some embodiments, the gap has a duration such that the total cycle time, corresponding to the time of the series of pulses plus the gap without electrical stimulation, equals a desired total duration. In some embodiments, the desired total duration is at least 0.6 (e.g., at least 6, at least 60) seconds and at most 120 (e.g., at most 60, at most 6) seconds. In some embodiments, the duration of the period of pulses is at least 0.6 (e.g., at least 6, at least 60) seconds and at most 120 (e.g., at most 60, at most 6) seconds. In some embodiments, the duration of the gap without electrical stimulation is at least 0.6 (e g., at least 6, at least 60) seconds and at most 120 (e.g., at most 119.4, at most 114, at most 60, at most 6) seconds. In some embodiments, the period of pulses contains at least 1 (e.g., at least 10, at least 12, at least 100, at least 120) and at most 1200 (e.g., at most 1000, at most 120) pulses. Without wishing to be bound by theory, it is believed that the gap without electrical stimulation can reduce (e.g., prevent) excess heating of at least one component of the bioreactor system. In some embodiments, the electric field within the hollow fiber cartridge 1200 is at least 1.46 (e.g., at least 5, at least 7.3) millivolts per millimeter (mV/mm) and at most 14.7 (e.g., at most 10, at most 7.3) mV/mm. In certain embodiments, the enclosure 1100 is connected to a ground of the function generator. In certain embodiments, the enclosure 1100 is at a voltage of 0 V. In certain embodiments, the conductor 1700 is mounted within the enclosure 1100 such that the conductor 1700 and the enclosure 1100 are not in electrical communication.

Figure 2 schematically depicts cross-sectional view of a computer-aided design model (“CAD model”) 2000 of an embodiment of the bioreactor system 1000. The CAD model 2000 depicts the enclosure 1100, the hollow fiber cartridge 1200 with the first inlet port 1202 and the first outlet port 1204, the conductor 1700, and the dielectric 1802/1804 that at least partially surrounds the hollow fiber cartridge 1200 disposed in the enclosure 1100.

Examples

Example 1 - Electric Field Modeling

The CAD model 2000 of Figure 2 was used to model the electric field inside the hollow fiber cartridge 1200. In the CAD model 2000, the hollow fiber cartridge 1200 contained Dulbecco's Modified Eagle Medium (“DMEM”) with a dielectric constant of 74.1 Farad per meter (F/m) and the dielectric 1802/1804 was Delrin®. The fibers inside the hollow fiber cartridge 1200 were ignored and set to the same dielectric constant as the DMEM. The enclosure 1100 was copper and the holes and gaps in the enclosure 1100 were ignored. The space within the enclosure 1100 was air and there was no air gap where the Delrin® dielectric 1802/1804 clamped the hollow fiber cartridge 1200. The voltage of the enclosure 1100 was 0 volts (“V”) and the voltage of the conductor 1700 was 3.4 V. The voltage on the conductor is variable and can be modified as needed for the specific requirements.

The modeling results are show in Figures 3a-c. Figure 3a shows a side view, Figure 3b shows a center cross-section view and Figure 3 c shows an end view at a side port of the hollow fiber cartridge 1100 of the bioreactor system 1000. At 3.4 V on the conductor 1700, the electric field strength in the central region of the hollow fiber cartridge 1100 was 5 millivolts per millimeter (mV/mm) ± 6% (ignoring low spots).

The electric field strength inside the hollow fiber cartridge 1100 of the bioreactor 1000 was linearly proportional to the voltage of the function generator attached to the conductor 1700. At a function generator voltage of 1 V, the electric field strength was 1.46 mV/mm in the center area of the hollow fiber cartridge 1100. At a function generator voltage of 3.4 V, the electric field strength was 5 mV/mm in the center area of the hollow fiber cartridge 1100. At a function generator voltage of 5 V, the electric field strength was 7.3 mV/mm in the center area of the hollow fiber cartridge 1100. At a function generator voltage of 6.8 V, the electric field strength was 10 mV/mm in the center area of the hollow fiber cartridge 1100. At a function generator voltage of 10 V, the electric field strength was 14.7 mV/mm in the center area of the hollow fiber cartridge 1100. Error bars for the electric field strength were 6%. A relationship of y = 1 ,4712x - 0.0172 to relate the electric field strength in to the function generator voltage was determined, where x is the function generator voltage in V and y is the electric field strength in mV/mm.

Example 2 - Electrical Stimulation of Cells

Cells were grown in the bioreactor system 1000 under different electrical stimulation conditions to monitor the effect of different electrical stimulation parameters on the extracellular vesicle cargo quantity, extracellular vesicle size and extracellular vesicle quantity.

Figure 4 shows a schematic of an example electrical stimulation and extracellular vesicle (“EV”) extraction procedure. For a week prior to cell introduction, the hollow fiber cartridge 1100 was washed with the growth medium DMEM by circulating growth media from the fluid reservoir 1400 through the first inlet port 1202 and out the first outlet port 1204 of the hollow fiber cartridge 1100 back to the fluid reservoir 1400 using the pump 1500. On day 0, the hollow fiber cartridge 1100 of the bioreactor system 1000 was seeded with HT1080 cells by injecting 2 x 10⁸ cells in growth media through the second inlet port 1206. Electrical stimulation began on day 2 with EV collection on days 2, 4, 7, 9 and 11. For EV collection, 20 ml was removed from the second outlet port 1208. Growth media was cycled through the first inlet port 1202 and first outlet port 1204 of the hollow fiber cartridge 1100 for the duration of the cell growth period.

Figure 5 shows a schematic of the electrical stimulation modalities used. A square wave pulse with a duration of 500 microseconds (ps) and a voltage of 4 V was generated by the function generator. The voltage created a field strength of approximately 5 mV/mm in the center of the hollow fiber cartridge 1100. In the 2 hertz (“Hz”) electrical stimulation modality, 120 pulses were generated in 60 seconds followed by a gap without electrical stimulation of 60 seconds. In the 20 Hz electrical stimulation modality, 120 pulses were generated in 6 seconds followed by a gap without electrical stimulation of 114 seconds. In the 200 Hz electrical stimulation modality, 120 pulses were generated in 0.6 seconds followed by a gap without electrical stimulation of 119.4 seconds. In each electrical stimulation modality, the duration of the gap was selected such that the total cycle time, corresponding to the duration of the 120 pulses plus the gap without electrical simulation, was 120 seconds. The 120 second cycle was repeated for the duration of the electrical stimulation. Cells were grown without electrical stimulation within the enclosure 1100, without electrical stimulation outside of the enclosure 1100, and with electrical stimulation at 2 Hz, 20 Hz and 200 Hz within the enclosure.

Figure 6 shows UV absorption measurements on a Bio-rad LP liquid chromatograph system at 280 nm of the EV from each collection day to determine the amount of protein in the EV without electrical stimulation and with 2 Hz, 20 Hz and 200 Hz electrical stimulation. Figure 6 shows that the presence and frequency of the electrical stimulation pulses affected the amount of protein produced in the EV as measured by absorption at 280 nm.

The EV were treated with a fluorescent dye (calceinAM) in order to identify the EV related peak from a chromatographic spectrum. The EV were separated using size-exclusion chromatography with a flow rate at 0.5 ml/min.

The EV size was measured by light scattering and the amount of protein was determined by fluorescence detection of the fluorescent-labled antibody PD-L1 at Ex488/Em520.

Figure 7 shows two-dimensional (“2D”) histograms of the scattering versus fluorescence signal (top) and histograms of EV size as determined by scattering (bottom) for EV collected on day 9 from cells with no electrical stimulation with the enclosure (a), no electrical stimulation without the enclosure (b), 2 Hz electrical stimulation modality (c), 20 Hz electrical stimulation modality (d) and 200 Hz electrical stimulation modality (e). In the 2D histograms, the fluorescence signal below the blue horizontal line at ~ 30 was assigned as noise. Figure 7 shows that the frequency of electrical stimulation affected the amount of protein produced in the EVs, as measured by the fluorescence signal, the amount of the EV produced and the size distribution of the EV produced.

Example 3

Methods - Cell culture

Human fibrosarcoma cell line (HT1080) was cultured following the standard protocol with DMEM at 37° C with 5% CO2. The low glucose culture media included DMEM (1 g/L glucose, Gibco 11885-084, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Gibco 10437-028, Thermo Fisher Scientific) and 1% (v/v) Penicillin Streptomycin (Gibco 15140122, Thermo Fisher Scientific). The high glucose culture media included DMEM (4.5 g/L glucose, Gibco 11965-092, Thermo Fisher Scientific), 10% (v/v) CDM-HD serum replacement (FiberCell Systems Inc., New Market, MD) and 1% (v/v) Penicillin Streptomycin (Gibco 15140122).

Generation of cell conditioned media

A 3D cell culture system from FiberCell was used for all experiments. Each 3D cell cartridge (C2011, FiberCell Systems Inc.) was prepared according to the manufactory guideline through sequentially wash with PBS for 72 hours, DMEM + 1% Penicillin-Streptomycin for 28 hours and DMEM+10% FBS+1% Penicillin-Streptomycin for 44 hours. The collection duration was limited to 2 weeks. To maximize the efficiency of each cartridge, the required cell seeding number was doubled and each experiment was started with 2 x 10⁸ cells on day 1. Five sequential cell conditioned media samples were collected on days 3, 5, 8, 10 and 12 respectively. The electrical stimulation (ES) was started on day 3 right after the first conditional media was collected and stopped until day 12 after the fifth sample collection. The low glucose culture media was replaced by the high glucose culture media after the second sample was collected on day 5. The glucose level of the cell culture media was monitored daily using blood glucose meters (AimStrip Plus, Germaine laboratories, INC, San Antonio, TX; ACCU-CHEK Guide Me, Roche Diabetes Care, Inc., Indianapolis, IN). The temperature of cell media was monitored in each experiment using a thermometer (TENMA 72-7715, Newark, Chicago, IL). The difference in temperature of cell media were within 0.2 °C. for experiments.

Electrical stimulation

The electrical stimulation was applied through a custom-designed device that delivered uniformly distributed electromagnetic field. Wave signals were provided by a function generator (4055B, B&K Precision, Yorba Linda, CA). A square-wave pulse with 0.4 ms width and 4 V amplitude was applied that generated an electrical field strength of 5 mV/mm inside the 3D cell culture system according to a simulation performed with the QuickField 6.4 software (Tera Analysis Ltd, Columbia SC, USA). All stimulation protocols were designed in a repeated 2-min pattern that included a total of 120 pulses. For ES at 2 Hz, those 120 pulses were evenly delivered within the first minute and the second minute was left quiet. For ES at 20 Hz, 120 pulses were delivered within the first 6 seconds and the remaining 114 seconds were left quiet. Similarly, for ES at 200 Hz, 120 pulses were delivered within the first 0.6 seconds, and the other 119.4 seconds were left quiet. The long quiet periods were designed to avoid potential overheating caused by ES. An equal number of pulses were applied in each experiment to avoid potentially differences caused by any unknown energy transfer mechanism. The pulse sequences are shown in Figure 5.

EV purification

20 ml of cell conditioned media was first subjected to a low-speed centrifuged at 3000 xg (20 min, 4°C) with the supernatant filtered through a 0.22um syringe filter (Millex-GS, SLGSO33SS, MilliporeSigma, Burlington, MA). Then a 3-step protocol was used to purify EVs: 1) the sample volume was reduced using 10 KD Amicon™ Ultra-15 centrifugal filter units (UFC 901024, MilliporeSigma™); 2) most soluble proteins were removed with polyethylene glycol precipitation (PEG). A working solution of PEG was prepared with PEG8000 (Sigma Aldrich) at 24% with PBS and 75 mM NaCl. 0.5 ml of concentrated cell condition media was mixed with 250 pL of 24%-PEG (final concentration of 8%), stored at 4°C overnight and finally centrifuged at l,500x g (30 minutes at 4°C). The obtained pellets were dissolved in 500 pL calcium-free PBS. 3) Free polyethylene glycol and other remaining soluble proteins were removed using sizeexclusion chromatograph (SEC). 500 pL sample from step 2 was overlaid to a self-made SEC column (Bio-Rad Glass Econo-Column Chromatography Columns, 7371512, packed with 10 ml Sepharose CL-6B, 17016001, from Cytiva, Muskegon, MI). A low-pressure liquid chromatograph system (Biologic LP, Bio-Rad) was used to deliver a constant flowrate at 500 pL/min with 200 pL as the collecting fraction volume. Purified EVs were recovered in the first elution peak that included 5 fractions with top reading of protein level at 280 nm. 40 pL of elution from each of the 5 fractions were combined together and stored at -80 °C until proteomics analysis. Remaining purified EV samples were stored at 4 °C until nanoparticle flow cytometry analysis.

Nanoparticle flow cytometry analysis High sensitivity flow cytometry for nanoparticle analysis was performed using a Flow Nanoanalyzer (N30, NanoFCM, Xiamen, China). In details, 100 pL of isolated EV sample was mixed with 4 pL of Alexa488 anti-human TNFRSF10B antibody (FAB6311G, R&D Systems, Minneapolis, MN) and 2 pL of PE anti-human CD63 antibody (561925, BD Biosciences, Franklin Lakes, NJ) and incubated at 37 °C for 30 minutes. Unlabeled antibodies were removed using another SEC with the qEVsingle column (70 nm Gen 2, IZON, Medford, MA). Again, labeled EVs were recovered in the first elution peak and then subsequently subjected to nanoparticle data collections. All data were converted to FCS 3.0 using NF Profession (Version 1.0, NanoFCM) and analyzed with FlowJo (Version 10.8.1, BD, Franklin Lakes, NJ).

Mass spectrometry analysis

Mass spectrometry (MS) based proteomics of EV was performed. 200 pL of each isolated EV sample was first precipitated with cold acetone, then digested with trypsin and extracted using the S-Trap micro kit (ProtiFi, Fairport, NY). The concentration of peptide was determined using Pierce quantitative fluorometric peptide assay (Cat# 23290, Thermo Scientific, Rockford, IL). 11 - 18 pL of the peptides were analyzed through nano-ESI-LC/MS/MS using the Orbitrap Exploris mass spectrometer coupled to a Dionex nano-LC system (Thermo Scientific, Waltham, MA). Liquid chromatography (LC) was performed using multistep linear gradients with solvent A (2% acetonitrile and 0.2% formic acid in H2O) and solvent B (80% acetonitrile, 10% isopropyl alcohol, and 0.2% formic acid in H2O) under a constant flow rate of 300 nL/min. The mass spectrometer was set at a resolution of 60,000 (at 200 m/z) in data dependent acquisition, with a full MS I scan ranging from 340 to 1600 m/z. Dynamic exclusion was set to 25 seconds with cycle time in 3 seconds.

All raw MS files were analyzed using MaxQuant (Version 1.6.17.0). MS/MS spectra was set up to search against the SwissProt Human Database (2022 ver. 1), assuming trypsin digestion with up to two missed cleavages with the fragment ion tolerance of 20 PPM and parent ion tolerance of 4.5 ppm. Cysteine carbamidomethylation was set as a fixed modification and methionine oxidation was set as a variable modification. The false discovery rate was set to 0.01 for protein level and peptide spectrum match. Protein identification required at least one unique or razor peptide per protein group. Only proteins with unique peptide were used in further data analysis. Contaminants, and reverse identification were excluded from further data analysis. Relative intensity based absolute quantification (riBAQ) was used to determine the relative molar abundances of proteins, which normalized each protein’ s iB AQ value to the sum of all iBAQ values from that sample.

MATLAB was used to generate a heat map and the relative difference was used to show effects of ES on the abundance of protein in EVs. Calculations of relative differences (5) for each identified protein (k) were performed using the following equation.

For each experimental condition, only the middle three collected samples, namely 2, 3, and 4 representing samples collected on day 5, day 8 and day 10, respectively, were used. The y- axis in the heat map was 8, and x-axis was experimental conditions.

To determine whether the protein has been identified as a membrane protein or existing in extracellular exosome, a custom code in MATLAB (The MathWorks Inc (2020). MATLAB Version: 9.8.0 (R2020a)) was used to search against the SwissProt human database (downloaded in March 2023, 69670 entries; 2118 entries).

Statistical evaluations

To determine the significance of differences in the percentage of TNFRSF10B/CD63 positive EV between experimental conditions, we performed a two-sample Kolmogorov- Smirnov test (KS-test) with weight using the Scipy package in Python. The weight was determined using exponential smoothing methods. Other codes were all written in MATLAB.

Results

Three rounds of experiments were performed with each round containing four different conditions including the control condition without electrical stimulation (No-ES) and three with ES at different frequencies of 2 Hz (low), 20 Hz (intermediate) and 200 Hz (high). EVs collected from the first round were used for protocol optimization where Calcein-AM was added to distinguish intact EVs from other nanoparticles or soluble proteins. Fluorescence-enhanced SEC was used to make certain that the first elution peak of UV absorbance at 280 nm superimposed with the unique peak of the fluorescent signal for the EV population, so that the UV signal alone could be used to determine the EV elution. Since the fluorescence of Calcein could contaminate the mass spectrometry and high-resolution nanoflow cytometry analyses, in the 2^nd and 3 ^rd rounds of EV collections Calcein was not added and the samples were subjected to all downstream analysis.

Twenty-four samples (two rounds, four conditions with three samples per condition) were submitted for mass spectrometry data analysis (see Methods above for details). In the last batch of samples, a total number of 2,078 proteins was identified, 732 proteins were present in all samples, which were used in the analysis.

To describe how EMF affects EV protein expression, the relative difference (5) algorithm was used that calculated changes on consecutively collected samples from the same experimental condition (see Methods above for details). A positive 5 value means increased protein expression while a negative 6 value means decreased protein expression under the corresponding condition.

For the control condition without ES, 176 (24%) of 732 identified proteins had increased expression; 214 (29%) for ES at 2 Hz, 158 (22%) for ES at 20 Hz and a sizable 422 (58%) for ES at 200 Hz (figure 8). It is worth noting the significantly large percentage of increased protein expression under ES at 200 Hz. Figure 8 is the heat map of 732 proteins.

To gain an in-depth understanding of the biological meaning of these proteins, enrichment analysis was performed based on previously published results using DAVID (the database for annotation, visualization, and integrated discovery) to extract biological features associated with the list of 732 protein genes. 115 types of biological processes were found to be associated with 456 genes.

Graph neural network (GNN) that connects extracted biological features to associated proteins and their calculated 5 values was used to determine how EMF could affect biological features. A compact list that included top ten candidates with the most positive 5 value and ten others with the most negative 5 value for each experimental condition. After taking out twenty- one duplicates, the final concise list contained 59 proteins (see Table 1), which was used to illustrates how EMF affects biological features. There were 37 types of biological processes associated with 33 proteins. To improve visualization, biological processes connected with two or more associated proteins were included. Table 1

Figures 9a-9d show four GNNs representing data from four different experimental conditions. The larger circles represent identified biological processes, whereas the smaller circles represent proteins with positive or negative 5. The sizes of the larger circles correspond to the number of associated proteins, while the sizes of the smaller circles represent the corresponding absolute 5 values.

GNN is a class of artificial neural networks commonly used for processing data that connects objects with edges. It can be observed that some biological features were enhanced at a specific frequency. For example, at the lower-right corner is a subgroup network for mRNA processing/splicing that associated with two proteins (PRPF8 and HNRNPC). Significant increases in both proteins were observed under ES at 200 Hz, suggesting that mRNA activities can be enhanced by ES at a high frequency. Significant increases S100A9, that has associations with four biological features including immunity, innate immunity, inflammatory response, and apoptosis, was observed under ES at 2 Hz. TNFRSF10B, that also associates with apoptosis, had its biggest increase under ES at 2 Hz, suggesting that immunity/apoptosis could be enhanced by ES at a low frequency.

GNN was used to illustrate how EMF affects biological features through modulations of EVs and found that different biological features respond to different frequencies. For example, a significantly elevated percentage of increased protein expression was observed at a 200 Hz frequency. Specifically, two proteins (PRPF8 and HNRNPC) associated with mRNA processing/splicing were elevated at 200 Hz, whereas the protein of S1009A associated with immunity/apoptosis was increased at 2 Hz.

Without wishing to be bound by theory, it is believed that mass spectrometry data represents total exosomal proteins regardless of whether the identified protein is from cytosolic origin or others located on EV surface. Identifying exosomal surface proteins can be important as they carry information on their tissues of origin and play a role as mediators for intercellular communications.

In flow cytometry, a fluorescent-tagged antibody specifically targeting the extramembrane domain of a surface protein was used. The selection of target was based on the criteria that: 1) it must be an exosomal surface protein; 2) it had a higher fraction among all detected proteins; 3) it is a well- studied protein with biological relevance; 4) it has a commercially available antibody suitable for flow cytometry. As a result, the TNFRSF10B (Tumor Necrosis Factor receptor superfamily member 10b, also known as the death receptor 5, DR5, or the TRAIL receptor 2, TRAILR2) was chosen as the evaluation target. TNFRSF10B contains an intracellular death domain, which can be activated by TNF -related apoptosis inducing ligands and transduces an apoptosis signal. Another target protein is the exosomal marker CD63. An Alexa488-tagged TNFRSF10B antibody and a PE-tagged CD63 antibody were used for duo-labeling and single-vesicle characterization was performed on a nanoflow cytometry analyzer (FCM N30).

Figures lOa-lOd include four scatter plots showing distributions of EVs detected from flowcytometry with the excitation wavelength at 488 nm. The Alexa488 signal was represented by the vertical axis and the PE signal was indicated in the horizontal axis. In each scatter plot, the distribution was convened into four quarters based on the baseline cutoff of the green and red fluorescent signals. On the top-left corner (QI) was the population for TNFRSF10B+ only EVs. Following clockwise on the right-top (Q2) was the population of EVs with duo-labeling of TNFRSF10B+/CD63+ EVs, and on the lower right (Q3) was the population for CD63+ only EVs. Q4 on the lower left was the distribution of non-specific EVs or other nano particles are not of interest.

To illustrate changes of each distribution, a bar graph was generated (Figure 11), in which the height of each bar indicates the percentage (P) for each distribution (PQI, PQ2 and PQ3). Samples from the 2^nd, 3^rd and 4^th collections are indicated in sequence respectively. The four different experimental conditions were labeled as No-ES, ES at 2 Hz, 20 Hz and 200 Hz, respectively.

For QI, the PQI of 2^nd collections from all four conditions were around 1.5-2 %. For ES at 2 Hz, however, PQI in the 3^rd and 4^th collections increased to 7-8% (-4 fold). PQ2 had a similar increase from 2^nd to 3^rd or 4^th collections when compared with that of No-ES or ES at 200 Hz (p < 0.05). Interestingly, PQ3 with ES at 20 Hz was lower than any other three conditions (p < 0.05). In conclusion, ES at 2 Hz selectively increased the percentage of TNFRSF10B+ EVs, but ES at 20 Hz reduced percentage of CD63+ EVs. Differences in the average size (0) of four EV distributions were also detected (0QI =84.5 nm ±6.5 nm, 0 Q2 =95.4nm±6.3nm, 0 Q3 =74.4nm±3.4nm, 0 Q4 =75.1nm±3.2nm). However, no detectable changes were found in the average size of each distribution affected by ES at any three frequencies.

The results demonstrate the complexity of EVs and show that caution should be applied when using commonly recognized EV makers (e.g., CD9, CD63 and CD81) to describe all EVs. Here, CD63 only represented a certain percentage among all EVs. Similarly, TNFRSF10B has an even lower percentage. Clearly, the subgroup of TNFRSF10B+ only EVs is very different from the subgroup of CD63+ only EVs, as indicated by their size measurements through side scattered light. The TNFRSF10B+/CD63+ dual-labeled subgroup had the largest average size, followed by the TNFRSFlOB-only labeled subgroup and the CD63-only subgroup. There is a possibility that the measured average size could be artificially expanded due to attached antibodies when the size of EV is on a similar scale to an antibody-tag complex. EMF induced changes in average size of each subgroup were not observed.

Interestingly, it was observed that at a lower frequency of 2 Hz, TNFRSF10B+ subgroups increased by 3~4 fold. TNFRSF10B is mainly located on plasma membrane. As an important mediator of the extrinsic pathways of apoptosis, therapies targeting TNFRSF10B quickly emerged as cancer treatments that utilize two type of pharmaceutical agents, recombinant human TRAIL proteins (such as Dulanermin) and TRAFL-R2 agonist antibodies (such as conatumumab, lexatumumab, tigatuzumab and drozitumab).

Although the preclinical results for TRAIL-R2 agonist antibodies were promising, the response rate or recovery rate was low when they were tested in patients. A recent study suggested that the abundance of TNFRSF10B on surface of cell could be regulated by vesicle transport and the higher expression of TNFRSF10B were correlated with higher first progression survival and post-progression survival in chemotherapy-treated lung cancer patients.

The results of increased TNFRSF10B+ EVs would be an indication of increased TNFRSF10B protein expression on cell surface. Thus, the combination of chemotherapy with EMF enhancement could be beneficial to those patients who have decreased response to initial TRAIL-R2 treatment. Without wishing to be bound by theory, it is believed that the methods can be relatively easily adopted to large-scale productions of EVs.

While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described herein as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

Particular embodiments of the subject matter have been described. Other embodiments are within the scope of the following claims. For example, the actions recited in the claims can be performed in a different order and still achieve desirable results. As one example, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In certain implementations, multitasking and parallel processing may be advantageous.

Claims

1. A bioreactor system comprising: an enclosure defining an interior, the enclosure configured to block at least some externally originating electromagnetic radiation from reaching the interior; a conductor disposed within the interior of the enclosure and configured to emit an electromagnetic field; a hollow fiber cartridge disposed within the interior of the enclosure and comprising: a housing defining an internal space; a plurality of hollow fibers within the internal space, each of the hollow fibers defining a lumen; a first inlet port and a first outlet port that are each in fluid communication with the lumens of the plurality of hollow fibers; and a second inlet port and a second outlet port that are each in fluid communication with the internal space of the housing that is external of the plurality of hollow fibers; a reservoir configured to contain a fluid; a pump configured to propel the fluid from the reservoir through the lumens of the plurality of hollow fibers via the first inlet port and the first outlet port; and a signal generator configured to deliver pulses of electrical energy to the conductor.

2. The bioreactor system of claim 1, further comprising a dielectric material; wherein the dielectric material is disposed within the interior of the enclosure; and wherein the dielectric material surrounds at least a portion of the hollow fiber cartridge.

3. The bioreactor system of claim 1 or 2, wherein the enclosure is connected to a ground of the signal generator.

4. The bioreactor system of any one of claims 1 to 3, further comprising tubing, wherein the tubing permits fluid communication between the first inlet port and first outlet port of the hollow fiber cartridge and the reservoir.

5. The bioreactor system of claim 4, wherein the tubing connects: (i) the reservoir and the pump and (ii) the pump and the first inlet port of the hollow fiber cartridge.

6. The bioreactor system of claim 4 or 5, wherein the tubing connects the first outlet port of the hollow fiber cartridge and the reservoir.

7. The bioreactor system of any one of claims 1 to 6, wherein the conductor is mounted to an interior surface of the enclosure, and wherein the conductor and the enclosure are electrically isolated from each other.

8. The bioreactor system of any one of claims 1 to 7, wherein the enclosure defines ports, wherein the ports enable fluid communication of at least one member selected from the group consisting of the first inlet port, the first outlet port, the second inlet port and the second outlet port with a component external to the enclosure.

9. The bioreactor system of claim 8, wherein the component external to the enclosure comprises at least one member selected from the group consisting of the reservoir, the pump, a tube, and a syringe.

10. The bioreactor system of claim 2, wherein the dielectric material comprises a polymer.

11. The bioreactor system of claim 2 or 10, wherein the dielectric material comprises polyoxymethylene or Delrin®.

12. The bioreactor system of any one of claims 1 to 11, wherein the enclosure comprises copper.

13. The bioreactor system of any one of claims 1 to 12, wherein the conductor comprises copper.

14. A method of increasing or decreasing an amount of one or more cargo molecules present in or on a surface of an extracellular vesicle derived from a target cell using the bioreactor system of any one of claims 1-13, the method comprising; electrically stimulating the target cell, wherein the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers; and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced without electrical stimulation.

15. A method of increasing or decreasing an amount of extracellular vesicles derived from a target cell using the bioreactor system of any one of claims 1-13, comprising; electrically stimulating the target cell, wherein the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers; and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced without electrical stimulation.

16. A method of increasing or decreasing a size of extracellular vesicles derived from a target cell using the bioreactor system of any one of claims 1-13, comprising; electrically stimulating the target cell, wherein the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers; and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced without electrical stimulation.

17. The method of any one of claims 14-16 wherein the electrical stimulation comprises an electric field of from 1 mV/mm to 10 mV/mm in the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers.

18. The method of any one of claims 14-16 wherein the electrical stimulation comprises an electric field of from 4 mV/mm to 6 mV/mm in the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers.

19. The method of any one of claims 14-16, wherein, in order to cause the electrically stimulation of the target cell, the signal generator generates pulses with a frequency of from 2 Hz to 200 Hz.

20. The method of claim 19, wherein the pulses are generated for a period of from 0.6 seconds to 60 seconds followed by a gap without pulses of from 0.6 seconds to 120 seconds.

21. The method of claim 20, wherein the pulse period and the gap are alternated repeatedly.

22. A method of increasing or decreasing an amount of one or more cargo molecules present in or on a surface of an extracellular vesicle derived from a target cell using the bioreactor system of any one of claims 1-13, comprising; disposing the target cell within the enclosure; wherein the target cell is further disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers; and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced in the absence of the enclosure.

23. A method of increasing or decreasing an amount of extracellular vesicles derived from a target cell using the bioreactor system of any one of claims 1-13, comprising; disposing the target cell within the enclosure; wherein the target cell is further disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers; and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced in the absence of the enclosure.

24. A method of increasing or decreasing a size of extracellular vesicles derived from a target cell using the bioreactor system of any one of claims 1-13, comprising; disposing the target cell within the enclosure; wherein the target cell is further disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers; and wherein the amount of the one or more cargo molecules is increased or decreased as compared to the amount of the one or more cargo molecules present inside or on the surface of an extracellular vesicle of the same or same type of target cell produced in the absence of the enclosure.

25. The method of any one of claims 15-16 or 23-24, wherein the extracellular vesicle derived comprise one or more cargo molecules present in or on a surface of the extracellular vesicles.

26. The method of claims 14, 22 or 25, wherein the cargo molecule is a protein.

27. The method of claim 14, 22 or 25, wherein the cargo molecule is a nucleic acid.

28. The method of claim 27, wherein the nucleic acid is a miRNA.

29. The method of any one of claims 14-28, wherein the target cell is disposed within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers by injection through the second inlet port.

30. The method of any one of claims 14-29, wherein the extracellular vesicles are removed from within the internal space of the housing of the hollow fiber cartridge that is external of the plurality of hollow fibers through the second outlet port.

31. The method of any one of claims 14-30, wherein the fluid is a cell growth medium.