WO2015060979A1

WO2015060979A1 - Bioreactor and perfusion system

Info

Publication number: WO2015060979A1
Application number: PCT/US2014/057259
Authority: WO
Inventors: Kayvan R. KESHARI; Mark VAN CRIEKINGE
Original assignee: The Regents Of The University Of California
Priority date: 2013-10-24
Filing date: 2014-09-24
Publication date: 2015-04-30

Abstract

Bioreactor constructs and related bioreactor perfusion systems find use in the growth and/or monitoring of living cells and/or tissues. Methods of using the bioreactor constructs and bioreactor perfusion systems are also provided. The disclosed constructs and bioreactor perfusion systems find particular use in connection with the analysis of cells and/or tissues via nuclear magnetic resonance (NMR) spectroscopy.

Description

BLOREACTOR AND PERFUSION SYSTEM

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/895,306, filed October 24, 2013, which application is incorporated herein by reference in its entirety and for all purposes.

INTRODUCTION

[0002] Changes in the metabolism of a biological system can imply progression to a given disease state. Aberrations in the metabolism of substrates, such as glucose and glutamine, have been linked to not only cancer, but also a wide range of other diseases and microenvironments. Thus, there has been a renewed interest in the field of metabolism, both at steady state and in dynamic flux, well downstream of their genetics and proteomics. Unlike genetics and proteomics, small changes in environment, such as pH, oxygenation and substrate concentrations can lead to instantaneous changes in metabolism. With this in mind, metabolism has been leveraged for the development of clinically relevant biomarkers for disease and response to therapy.

[0003] With the increasing interest in metabolism, has come the need for relevant platforms to facilitate non-invasive biochemical study and biomarker translation. These systems, deemed bioreactors, provide an environment where cells and tissues can grow or change with control of the environment and intervention. Bioreactors geared toward metabolic investigation, have typically been used on the bench top in conjunction with methods to measure distributions of metabolites and tracers, most of which are destructive (e.g. extraction and HPLC, NMR and/or MS). Non-invasive optical methods have also been used to characterize these systems, but they have limited translation to the clinical setting. Moreover, they are not well suited for small densities of cells and tissues.

[0004] Non-destructive methods have been employed to observe changes in

bioreactor systems in time, most often through the use of magnetic resonance (MR) spectroscopy and imaging. Development of MR-compatible bioreactor systems for the study of cell metabolism non-invasively has been limited though by the sensitivity of low γ nuclei, such as 13 C and 15 N. With the recent work in dissolution dynamic nuclear polarization (DNP) technology, the requirements for cell density in MR-compatible bioreactors have been relaxed given the dramatic enhancement in SNR achieved. Hyperpolarized substrates, injected into living systems, provide a means for studying rapid dynamic processes and are a new direction in the development of translatable non-invasive biomarkers. Typical studies in traditional

10 mm MR-compatible culture systems or larger still require on the order of 10 cells, which is very difficult to achieve in primary cell and tissue cultures. The adaptation of this technology to smaller cultures (e.g., on the order of 5 x 10⁶ cells) is non-trivial and may include the modification of both engineering parameters (e.g., flow rate, MR-compatible mechanical structure) and biochemical variables (e.g., dissolved oxygen, real time concentration of glucose). The engineering of more compact systems would allow for the study of primary cultures of cells and tissues, which are more clinically relevant and cost efficient. This technology could then be extended to use in multiple systems and adapted for other non-invasive monitoring approaches.

SUMMARY

[0005] The present disclosure provides bioreactor constructs and related bioreactor perfusion systems which find use, for example, in the growth and/or monitoring of living cells and/or tissues. Related methods of using the disclosed bioreactor constructs and bioreactor perfusion systems are also provided. In some embodiments, the disclosed bioreactor constructs and bioreactor perfusion systems find particular use in connection with the analysis of cells and/or tissues via nuclear magnetic resonance (NMR) spectroscopy.

[0006] Certain non-limiting aspects of the disclosure are provided below:

1. A bioreactor system including:

a concentric exchanger including

a first elongate structure defining a first bore extending lengthwise therethrough,

a second elongate structure defining a second bore extending lengthwise therethrough, and

a third elongate structure defining a third bore extending lengthwise therethrough, wherein the first elongate structure is positioned concentrically within the second bore of the second elongate structure and the second elongate structure is positioned concentrically within the third bore of the third elongate structure; and

a bioreactor coupled to the concentric exchanger and configured to fluidically receive an input from the first bore of the first elongate structure.

The bioreactor system of 1 , wherein the third elongate structure includes an external insulation layer.

The bioreactor system of 1 or 2, wherein the third elongate structure is fluidically coupled to a temperature-controlled fluid flow.

The bioreactor system of 3, wherein the temperature-controlled fluid flow is a temperature-controlled water flow.

The bioreactor system of any one of 1 to 4, wherein the second elongate structure is fluidically coupled to a pressurized gas input.

The bioreactor system of any one of 1 to 5, wherein the first elongate structure is fluidically coupled to a perfusion media input.

The bioreactor system of any one of 1 to 6, wherein one or more of the first, second and third elongate structures are tubular structures.

The bioreactor system of any one of 1 to 7, wherein the third elongate structure includes a flexible polymer material.

The bioreactor system of 8, wherein the third elongate structure includes an external insulation layer.

The bioreactor system of any one of 1 to 9, wherein the second elongate structure includes a flexible polymer material.

The bioreactor system of 10, wherein the flexible polymer material includes one or more of a fluorinated ethylene propylene (FEP), a polytetraf uoroethylene (PTFE), and a perf uoroalkoxy (PFA).

The bioreactor system of 10, wherein the flexible polymer material is relatively gas impermeable.

The bioreactor system of any one of 1 to 12, wherein the first elongate structure includes a flexible polymer material.

The bioreactor system of 13, wherein the flexible polymer materials is relatively gas permeable.

The bioreactor system of 13, wherein the flexible polymer material includes a silicone. The bioreactor system of any one of 1 to 15, wherein the bioreactor includes a bioreactor construct including a UV-cured polymer material.

The bioreactor system of any one of 1 to 16, wherein the bioreactor includes a bioreactor construct including a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

The bioreactor system of 17, wherein the bioreactor construct includes a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water. The bioreactor system of any one of 1 to 16, wherein the bioreactor includes a bioreactor construct including a magnetic resonance (MR) compatible material. The bioreactor system of any one of 1 to 19, wherein the bioreactor includes a bioreactor construct sized to fit within an approximately 5 mm outer diameter tube. The bioreactor system of any one of 1 to 20, wherein the bioreactor includes a bioreactor construct sized to fit within an approximately 4 mm inner diameter tube. The bioreactor system of any one of 1 to 20, wherein the bioreactor includes a bioreactor construct having a diameter of about 4 mm.

The bioreactor system of any one of 1 to 20, wherein the bioreactor includes a bioreactor construct having a diameter of less than 4 mm.

The bioreactor system of any one of 1 to 23, wherein the bioreactor includes a bioreactor construct including a first end and a second end and defines a central bore extending from the first end to the second end, wherein the central bore is configured to fluidically receive the input from the first bore of the first elongate structure.

The bioreactor system of 24, wherein the central bore has a diameter of from about 1 mm to about 2 mm.

The bioreactor system of 24, wherein the central bore has a diameter of from about 0.5 mm to about 1 mm.

The bioreactor system of 24 or 25, wherein the bioreactor construct includes a central shaft defining the central bore.

The bioreactor system of 27, wherein the bioreactor construct includes a plurality of sidewalls extending the length of the bioreactor construct and defining sidewall openings in the bioreactor construct.

The bioreactor system of 28, wherein the bioreactor construct includes a plurality of struts extending perpendicularly from the central shaft and connecting the central shaft with the plurality of sidewalls. The bioreactor system of any one of 27 to 29, wherein the first end and the second end of the bioreactor construct each define a plurality of openings which, when the bioreactor construct is positioned in a tube, provide a flow path between the first end, the sidewall openings and the second end.

The bioreactor system of any one of 27 to 30, wherein the diameter of the central bore is greater at the first end of the bioreactor construct and the second end of the bioreactor construct than at a midpoint along the central bore.

The bioreactor system of any one of 27 to 31 , wherein the first end and the second end of the bioreactor construct each include a plurality of ridges extending at least partially along the length of the bioreactor construct.

The bioreactor system of 24 or 25, wherein the bioreactor construct defines a plurality of through holes positioned externally to the central bore and extending from the first end to the second end.

The bioreactor system of 33, wherein the through holes are positioned radially around the central bore.

The bioreactor system of 33 or 34, wherein the through holes extend parallel to the central bore.

The bioreactor system of any one of 33 to 35, wherein the through holes have a diameter of from about 0.2 mm to about 0.6 mm.

The bioreactor system of 36, wherein the through holes have a diameter of about 0.4 mm.

The bioreactor system of 36, wherein the through holes have a diameter of about 300 μιη.

The bioreactor system of any one of 33 to 37, wherein the length of the bioreactor construct from the first end to the second end is from about 2 mm to about 6 mm. The bioreactor system of 39, wherein the length of the bioreactor construct from the first end to the second end is about 3 mm.

The bioreactor system of any one of 1 to 39, including a first manifold coupled to the concentric exchanger at a first end of the concentric exchanger, the first manifold including a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports, and wherein the first manifold provides a flow path between the third bore of the third elongate structure and the third port. The bioreactor system of 41 , wherein an external wall of the first port engages the third bore of the third elongate structure via press-fit engagement.

The bioreactor system of 42, wherein the external wall of the first port is sealed against the third bore of the third elongate structure with an adhesive.

The bioreactor system of 42, wherein the first port includes external barbs or ridges to effect the press-fit engagement.

The bioreactor system of any one of 41 to 44, including a seal positioned in the second port between the body of the manifold and the second elongate structure. The bioreactor system of any one of 41 to 45, including a second manifold coupled to the concentric exchanger at a second end of the concentric exchanger, the second manifold including a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port of the second manifold interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports of the second manifold, and wherein the second manifold provides a flow path between the third bore of the third elongate structure and the third port of the second manifold.

The bioreactor system of 46, wherein an external wall of the first port of the second manifold engages the third bore of the third elongate structure via press-fit engagement.

The bioreactor system of 47, wherein the external wall of the first port is sealed against the third bore of the third elongate structure with an adhesive.

The bioreactor system of 47, wherein the first port of the second manifold includes external barbs or ridges to effect the press-fit engagement.

The bioreactor system of any one of 46 to 49, including a seal positioned in the second port of the second manifold between the body of the second manifold and the second elongate structure.

The bioreactor system of any one of 41 to 50, wherein the first and/or second manifold includes a UV-cured polymer material.

The bioreactor system of any one of 41 to 51, wherein the first and/or second manifold includes a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

The bioreactor system of 52, wherein the first and/or second manifold includes a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water. The bioreactor system of any one of 41 to 51, wherein the first and/or second manifold includes a magnetic resonance (MR) compatible material.

The bioreactor system of any one of 46 to 54, wherein the third port of the first manifold functions as an inlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an outlet port for the temperature- controlled fluid flow.

The bioreactor system of any one of 46 to 54, wherein the third port of the first manifold functions as an outlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an inlet port for the temperature- controlled fluid flow.

The bioreactor system of any one of 1 to 56, including a perfusion media reservoir coupled to the first elongate structure.

The bioreactor system of 57, wherein the perfusion media reservoir receives perfusion media after it has passed through the bioreactor.

The bioreactor system of 57 or 58, including a pump configured to pump perfusion media from the perfusion media reservoir through the first bore of the first elongate structure.

The bioreactor system of 59, wherein the pump is a peristaltic pump.

The bioreactor system of any one of 1 to 60, including an injection port coupled to the first elongate structure.

The bioreactor system of any one of 1 to 61, including a pressurized gas reservoir coupled to the second elongate structure.

The bioreactor system of any one of 1 to 62, wherein the bioreactor is positioned in a tube.

The bioreactor system of 63, wherein the tube is an NMR tube.

The bioreactor system of any one of 1 to 64, including a tube cap, the tube cap including a body defining an inlet port, an outlet port, a tube-engagement opening, and an internal chamber connecting the inlet port, the outlet port and the tube engagement opening.

The bioreactor system of 65, wherein the body of the tube cap includes an internal wall at least partially defining the internal chamber, and wherein the internal wall defines a channel configured to facilitate liquid flow through the internal chamber and out the outlet port.

The bioreactor system of 66, wherein the channel has a spiral structure. 68. The bioreactor system of 65 to 67, wherein the tube cap includes a UV-cured polymer material.

69. The bioreactor system of any one of 65 to 68, wherein the tube cap includes a

material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

70. The bioreactor system of 69, wherein the tube cap includes a material having a

magnetic susceptibility within 5% of the magnetic susceptibility of water.

71. The bioreactor system of any one of 65 to 68, wherein the tube cap includes a

magnetic resonance (MR) compatible material.

72. The bioreactor system of any one of 65 to 71 , wherein the tube cap includes one or more O-rings positioned in the internal chamber so as to engage an external wall of a tube to which the tube cap is engaged.

73. The bioreactor system of any one of 65 to 72, including one or more connectors

which, individually or together, provide a fluid flow path from the first bore of the first elongate structure of the concentric exchanger through the inlet port of the tube cap to the bioreactor.

74. The bioreactor system of any one of 65 to 73, wherein the outlet port of the tube cap receives perfusion media after it has passed through the bioreactor.

75. The bioreactor system of 74, wherein the perfusion media reservoir is f uidically connected to the outlet port of the tube cap.

76. A bioreactor including:

a first end;

a second end;

a central shaft defining a central bore extending from the first end to the second end; and

a plurality of sidewalls extending the length of the bioreactor and defining sidewall openings in the bioreactor.

77. The bioreactor of 76, wherein the bioreactor includes a plurality of struts extending perpendicularly from the central shaft and connecting the central shaft with the plurality of sidewalls.

78. The bioreactor of 76 or 77, wherein the first end and the second end of the bioreactor each define a plurality of openings which, when the bioreactor is positioned in a tube, provide a flow path between the first end, the sidewall openings and the second end. 79. The bioreactor of any one of 76 to 78, wherein the diameter of the central bore is greater at the first end of the bioreactor and the second end of the bioreactor than at a midpoint along the central bore.

80. The bioreactor of any one of 76 to 79, wherein the first end and the second end of the bioreactor each include a plurality of ridges extending at least partially along the length of the bioreactor.

81. The bioreactor of any one of 76 to 80, wherein the bioreactor includes a UV-cured polymer material.

82. The bioreactor of any one of 76 to 81, wherein the bioreactor includes a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

83. The bioreactor of 82, wherein the bioreactor includes a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water.

84. The bioreactor of any one of 76 to 81, wherein the bioreactor includes a magnetic resonance (MR) compatible material.

85. The bioreactor of any one of 76 to 84, wherein the bioreactor is sized to fit within an approximately 5 mm outer diameter tube.

86. The bioreactor of any one of 76 to 85, wherein the bioreactor is sized to fit within an approximately 4 mm inner diameter tube.

87. The bioreactor of any one of 76 to 85, wherein the bioreactor has a diameter of about 4 mm.

88. The bioreactor of any one of 76 to 85, wherein the bioreactor has a diameter of less than 4 mm.

89. A concentric exchanger including:

a first elongate structure defining a first bore extending lengthwise therethrough;

a second elongate structure defining a second bore extending lengthwise therethrough;

a third elongate structure defining a third bore extending lengthwise therethrough, wherein the first elongate structure is positioned concentrically within the second bore of the second elongate structure and the second elongate structure is positioned concentrically within the third bore of the third elongate structure; and a first manifold coupled to the concentric exchanger at a first end of the concentric exchanger, the first manifold including a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports, and wherein the first manifold provides a flow path between the third bore of the third elongate structure and the third port.

90. The concentric exchanger of 89, wherein the third elongate structure includes an external insulation layer.

91. The concentric exchanger of 89 or 90, wherein the third elongate structure is

fluidically coupled to a temperature-controlled fluid flow.

92. The concentric exchanger of 91, wherein the temperature-controlled fluid flow is a temperature-controlled water flow.

93. The concentric exchanger of any one of 89to 92, wherein the second elongate

structure is fluidically coupled to a pressurized gas input.

94. The concentric exchanger of any one of 89to 93, wherein the first elongate structure is fluidically coupled to a perfusion media input.

95. The concentric exchanger of any one of 89 to 94, wherein one or more of the first, second and third elongate structures are tubular structures.

96. The concentric exchanger of any one of 89 to 95, wherein the third elongate structure includes a flexible polymer material.

97. The concentric exchanger of any one of 89 to 96, wherein the second elongate

structure includes a flexible polymer material.

98. The concentric exchanger of 97, wherein the flexible polymer material includes one or more of a fluorinated ethylene propylene (FEP), a polytetrafluoroethylene (PTFE), and a perfluoroalkoxy (PFA).

99. The concentric exchanger of any one of 89 to 98, wherein the first elongate structure includes a flexible polymer material.

100. The concentric exchanger of 99, wherein the flexible polymer material

includes a silicone.

101. The concentric exchanger of 100, wherein an external wall of the first port engages the third bore of the third elongate structure via press-fit engagement.

102. The concentric exchanger of 101 , wherein the first port includes external barbs or ridges to effect the press-fit engagement.

103. The concentric exchanger of any one of 89 to 102, including a seal positioned in the second port between the body of the manifold and the second elongate structure. 104. The concentric exchanger of any one of 89 to 103, including a second manifold coupled to the concentric exchanger at a second end of the concentric exchanger, the second manifold including a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port of the second manifold interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports of the second manifold, and wherein the second manifold provides a flow path between the third bore of the third elongate structure and the third port of the second manifold.

105. The concentric exchanger of 104, wherein an external wall of the first port of the second manifold engages the third bore of the third elongate structure via press-fit engagement.

106. The concentric exchanger of 105, wherein the external wall of the first port of the second manifold is sealed against the third bore of the third elongate structure with an adhesive.

107. The concentric exchanger of 105, wherein the first port of the second

manifold includes external barbs or ridges to effect the press-fit engagement.

108. The concentric exchanger of any one of 104 to 107, including a seal

positioned in the second port of the second manifold between the body of the second manifold and the second elongate structure.

109. The concentric exchanger of any one of 89 to 108, wherein the first and/or second manifold includes a UV-cured polymer material.

110. The concentric exchanger of any one of 89 to 109, wherein the first and/or second manifold includes a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

111. The concentric exchanger of 1 10, wherein the first and/or second manifold includes a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water.

112. The concentric exchanger of any one of 89 to 109, wherein the first and/or second manifold includes a magnetic resonance (MR) compatible material.

113. The concentric exchanger of any one of 104 to 112, wherein the third port of the first manifold functions as an inlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an outlet port for the temperature-controlled fluid flow. 114. The concentric exchanger of any one of 104 to 112, wherein the third port of the first manifold functions as an outlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an inlet port for the temperature-controlled fluid flow.

115. The concentric exchanger of any one of 89 to 114, including a perfusion

media reservoir coupled to the first elongate structure.

116. The concentric exchanger of 1 15, including a pump configured to pump

perfusion media from the perfusion media reservoir through the first bore of the first elongate structure.

117. The concentric exchanger of 1 16, wherein the pump is a peristaltic pump.

118. The concentric exchanger of any one of 89 to 117, including an injection port coupled to the first elongate structure.

119. The concentric exchanger of any one of 89 to 118, including a pressurized gas reservoir coupled to the second elongate structure.

120. A method of perfusing cells and/or tissues, the method including

positioning cells and/or tissues in a bioreactor of the bioreactor system of any one of 1-75; and

flowing media through the first bore of the first elongate structure of the concentric exchanger to the bioreactor, wherein the cells and/or tissues in the bioreactor are perfused by the media.

121. The method of 120, wherein the method further includes perfusing the cells and/or tissues while the bioreactor is positioned in an NMR tube in the bore of an NMR spectrometer.

122. The method of 121 , wherein the method further includes analyzing the cells and/or tissues using NMR spectroscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 provides a schematic of a closed-loop bioreactor perfusion system including a bioreactor, a concentric exchanger, and exchanger manifolds according to embodiments of the present disclosure.

[0008] Figure 2 provides a schematic of an open-loop bioreactor perfusion system, including a bioreactor, a concentric exchanger, and exchanger manifolds, according to embodiments of the present disclosure. [0009] Figure 3 shows a side view of a bioreactor perfusion system, including a spindle bioreactor construct, according to embodiments of the present disclosure.

[0010] Figures 4, 5 and 6 show a side view, cross-sectional view and perspective view, respectively, of a component of the bioreactor perfusion system depicted in Figure 3, including a cell/tissue culture tube, a spindle bioreactor construct, and a tube cap.

[0011] Figures 7, 8, 9, 10 and 11 show a perspective view, side view, first cross- sectional view, end view and second cross-sectional view, respectively, of a spindle bioreactor construct according to embodiments of the present disclosure.

[0012] Figure 12 shows a side view of a bioreactor perfusion system, including a fret bioreactor construct, according to embodiments of the present disclosure.

[0013] Figures 13, 14 and 15 show a side view, cross-sectional view and

perspective view, respectively, of a component of the bioreactor perfusion system depicted in Figure 12, including a cell/tissue culture tube, a fret bioreactor construct, and a tube cap.

[0014] Figures 16 and 17 show a perspective views and cross-sectional view,

respectively, of a fret bioreactor construct according to embodiments of the present disclosure.

[0015] Figures 18, 19, 20 and 21 show a cross-sectional view, side view with

interior portions shown in phantom, and two perspective views, respectively, of a tube cap according to embodiments of the present disclosure.

[0016] Figures 22, 23 and 24 show two perspective views and an end view,

respectively, of an exchanger manifold according to embodiments of the present disclosure.

[0017] Figures 25, 26, 27 and 28 show a side view, cross-sectional view and two perspective views, respectively, of a component of the bioreactor perfusion systems depicted in Figures 3 and 12, including an exchanger manifold connected to a three- tube concentric exchanger. Only a portion of the three-tube exchanger is shown for illustration purposes.

[0018] Figure 29 provides a schematic of a bioreactor perfusion system according to embodiments of the present disclosure (A); a schematic of a bioreactor perfusion system, including a bioreactor construct, according to embodiments of the present disclosure (B); and an MRI (magnetic resonance image) of an inlet tube showing flow pattern (C). [0019] Figure 30 shows a perspective view, a side view and a cross-sectional view of a three-tube concentric exchanger connected to an exchanger manifold according to embodiments of the present disclosure. Only a portion of the three-tube exchanger is shown for illustration purposes.

[0020] Figure 31 shows various views of an exchanger manifold designed to

interface with a three-tube concentric exchanger according to embodiments of the present disclosure.

[0021] Figure 32 shows various views of a fret bioreactor construct designed for the perfusion of suspended cells and/or encapsulates according to embodiments of the present disclosure.

[0022] Figure 33 shows various views of a spindle bioreactor construct designed for the perfusion of living primary tissues according to embodiments of the present disclosure.

[0023] Figure 34 shows various view of a tube cap designed to facilitate media delivery and removal and/or recirculation according to embodiments of the present disclosure.

[0024] Figure 35 provides an 1H spectrum acquired from bioreactor cultures.

Average 1H line-shape acquired from bioreactor cultures at 500 MHz (n = 7). The average characteristic of the peak shape were 10.9 ± 0.4 Hz (50% of peak max), 127.9 ± 3.7 Hz (0.55% of peak max) and 307.1 ± 15.7 Hz (0.11% of peak max).

[0025] Figure 36 provides images of representative histology of alginate

encapsulates post-24 hours of perfusion inside of a 5mm bioreactor. Hematoxylin and Eosin (H&E) demonstrate preserved cellular structure. Ki-67 nuclear antigen staining shows that greater than 95% cells are in a state of proliferation. Caspase-3 staining reaffirms that less than 5% of cells are apoptotic.

[0026] Figure 37 (Panel A) Biochemical Scheme of cellular metabolism when

13 18

utilizing hyperpolarized [1- C] pyruvate (HP Pyr) and F-deoxyglucose (FDG). (Panel B) Representative ³¹P NMR spectra of 15 x 10⁶ living PC-3 cells within the 5mm bioreactor. (Panel C) Average concentrations of observed metabolites (n = 6).

(Panel D) 13 C NMR spectra acquired on PC-3 cells post injection of 7mM HP Pyr. Time resolved data is shown with 3 sec resolution, acquired with a 10°pulse and acquire scheme. [0027] Figure 38 shows the results of injecting FDG into an empty chamber and the characteristic wash-in/out profile. Empty encapsulates do not retain the FDG tracer signal after wash-out.

[0028] Figure 39 (Panel A) Imaging of 4 bioreactors simultaneously with a wash-in of FDG of 40 min and analogous wash-out of 40 min. (Panel B) Plot of accumulated radioactivity after wash-out of the tracer, with a linear correlation (R =0.98) as expected for increasing cell density.

[0029] Figure 40 provides results for a demonstration of translatable biomarker response in PC-3 cells by NAMPT inhibition. (Panel A) Marked decrease in HP Lactate with treatment and increase in HP Alanine. (Panel B) Analogous decrease in FDG accumulation, indicative of changes in glycolysis. (Panel C) Gene expression of cells with treatment, reaffirming that the change is in the concentration of the cofactor NAD and not changes in expression of the relevant transporters and enzymes.

31 13

[0030] Figure 41 provides P and HP C MR spectra from living human prostate tissue slices. (Panel A) 3 IP spectra of 4 living tissue slices, with standard resonances readily visualized. (Panel B) HP lactate dynamics with a temporal resolution of 3

13

sees, post-injection of [1- C] pyruvate. (Panel C) Representative spectrum at 81 sees

13

after injection of [1,2- C] pyruvate. The CI of lactate, bicarbonate and C0₂ are derived from the CI of pyruvate, while the C5 glutamate resonance is derived from the C2 of pyruvate.

[0031] Figure 42 provides a perspective view of an embodiment of a horizontally stacked bioreactor construct according to the present disclosure.

[0032] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0033] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0034] Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0035] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a bioreactor construct" includes a plurality of such bioreactor constructs and reference to "the bioreactor perfusion system" includes reference to one or more bioreactor perfusion systems and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0036] To the extent the definition or usage of any term herein conflicts with a

definition or usage of a term in an application or reference incorporated by reference herein, the instant application shall control.

[0037] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0038] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. This is intended to provide support for all such combinations.

DETAILED DESCRIPTION

[0039] The present disclosure provides bioreactor constructs and related bioreactor perfusion systems which find use, for example, in the growth and/or monitoring of living cells and/or tissues. As used herein, the term "bioreactor" broadly refers to any apparatus in which a biological reaction or process is carried out.

[0040] Related methods of using the disclosed bioreactor constructs and bioreactor perfusion systems are also provided. The bioreactor constructs and bioreactor perfusion systems described herein allow biological processes of interest, e.g., metabolic processes, to be conducted and analyzed using any of a number of suitable analytic methods and/or tools, e.g., NMR spectroscopy and related devices, and positron emission tomography (PET) imaging and related devices.

Bioreactor Perfusion Systems

[0041] As discussed above, the present disclosure provides bioreactor perfusion systems. These systems may include closed-loop systems in which media, e.g., growth media, is recirculated through a bioreactor or open-loop systems in which media, e.g., growth media, flows through the bioreactor without recirculation.

[0042] For example, with reference to Fig. 1, the present disclosure provides a

closed-loop bioreactor perfusion system 100, including a bioreactor 102, a first exchanger manifold 104, a concentric exchanger 106, and a second exchanger manifold 108. The arrows in Fig. 1 depict the general direction of media flow in the system. Generally, media enters second exchanger manifold 108, which is coupled to concentric exchanger 106. As the media flows through concentric exchanger 106 it is equilibrated with respect to temperature and/or gas content. Equilibrated media then flows from concentric exchanger 106 into first exchanger manifold 104, wherein first exchanger manifold 104 is coupled to the concentric exchanger 106 at an opposite end of concentric exchanger 106 than second exchanger manifold 108. Equilibrated media then flows from first exchanger manifold 104 into bioreactor 102. After perfusing cells and/or tissue positioned in bioreactor 102, the media flows from bioreactor 102, through first exchanger manifold 104, and back into second exchanger manifold 108 (pathway A). Optionally, the system may be configures such that media flows from bioreactor 102, after perfusing cells and/or tissue, back into second exchanger manifold 108 without flowing back through first exchanger manifold 104 (pathway B). One or more reservoirs, pumps, ports and/or valves may be positioned at any suitable position in the system. For example, in some embodiments, one or more reservoirs, injection ports, and/or gas ports may be positioned upstream in the flow path from second exchanger manifold 108. Each of the bioreactor 102, concentric exchanger 106, and exchanger manifolds (104 and 108) are discussed in greater detail below along with additional optional elements of the described bioreactor perfusion system 100.

With reference to Fig. 2, the present disclosure provides an open-loop bioreactor perfusion system 200, including a bioreactor 202, a first exchanger manifold 204, a concentric exchanger 206, and a second exchanger manifold 208. The arrows in Fig. 2 depict the general direction of media flow (or optional media flow) in the system. Generally, media enters second exchanger manifold 208, which is coupled to concentric exchanger 206. As the media flows through concentric exchanger 206 it is equilibrated with respect to temperature and/or gas content. Equilibrated media then f ows from concentric exchanger 206 into first exchanger manifold 204, wherein first exchanger manifold 204 is coupled to the concentric exchanger 206 at an opposite end of concentric exchanger 206 than second exchanger manifold 208. Equilibrated media then flows from first exchanger manifold 204 into bioreactor 202. After perfusing cells and/or tissue positioned in bioreactor 202, the media flows from bioreactor 202, through first exchanger manifold 204, and out of the system, e.g., to a waste reservoir (pathway A).

Optionally, the system may be configured such that media flows from bioreactor 202 out of the system, e.g., to a waste reservoir, after perfusing cells and/or tissue, without flowing back through first exchanger manifold 204 (pathway B). One or more reservoirs, pumps, ports and/or valves may be positioned at any suitable position in the system. For example, in some embodiments, one or more reservoirs, injection ports, and/or gas ports may be positioned upstream in the flow path from second exchanger manifold 208. Each of the bioreactor 202, concentric exchanger 206, and exchanger manifolds (204 and 208) are discussed in greater detail below along with additional optional elements of the described bioreactor perfusion system 200. [0044] The bioreactor-perfusion systems disclosed herein may include one or more components including or prepared from magnetic resonance (MR) spectroscopy compatible materials and/or positron emission tomography (PET) compatible materials. Suitable materials may include, e.g., biologically inert polymers and/or UV-cured polymer materials. In some embodiments, one or more of the components of the bioreactor-perfusion systems disclosed herein may include material having a magnetic susceptibility within 10% of the magnetic susceptibility of water, such as a magnetic susceptibility within 5% of the magnetic susceptibility of water.

Bioreactor Perfusion System Including Spindle Bioreactor Construct

[0045] With reference to FIGS. 3 - 6, an embodiment of a bioreactor perfusion

system according to the present disclosure is provided. This embodiment may be either a closed loop bioreactor perfusion system 100 or an open loop bioreactor perfusion system 200 as described above. Bioreactor perfusion system 300 includes a spindle bioreactor construct 400 positioned in a cell and/or culture tube 304. A media delivery tube 344 is coupled to the spindle bioreactor construct 400 and configured to deliver media received from a concentric exchanger (106 or 206) to the spindle bioreactor construct 400. In bioreactor perfusion system 300, concentric exchanger (106 or 206) is a three-tube exchanger, including a first tube of the three-tube exchanger 326, a second tube of the three-tube exchanger 328, and a third tube of the three-tube exchanger 330. Bioreactor perfusion system 300 includes a tube cap 306 which engages cell and/or culture tube 304. An outlet tube 312 and outlet connector 314 are configured to allow a medium, which has flowed through spindle bioreactor construct 400 to exit cell and/or culture tube 304. An inlet tube seal 308 positioned at the top of tube cap 306 engages inlet tube 310 providing a fluid-tight seal. In some embodiments, media delivery tube 344 and inlet tube 310 are two portions of the same tube structure. In other embodiments, they may be distinct structures.

[0046] Inlet tube 310 connects to the first tube of the three-tube exchanger 326

through inlet connector 316, and first exchanger manifold connector 322. A second tube connector seal 346 engages the second tube of the three-tube exchanger 328, providing a fluid tight seal. The second tube of the three-tube exchanger 328 extends through first exchanger manifold 318, a first exchanger manifold seal 320 providing a fluid-tight seal therebetween. First exchanger manifold inlet/outlet tube 324 provides a means by which a fluid, e.g., a temperature controlled fluid such as water, may be flowed into a third tube of the three-tube exchanger 330. The third tube of the three-tube exchanger 330 engages a tube connecting flange 352 of the first exchanger manifold 318.

[0047] At the opposite end of concentric exchanger (106 or 206) the third tube of the three-tube exchanger 330 engages a tube connecting flange 352 of second exchanger manifold 332. The first tube of the three-tube exchanger 326 and the second tube of the three-tube exchanger 328 extend through second exchanger manifold 332. A second exchanger manifold inlet/outlet tube 334 provides a means by which a fluid, e.g., a temperature controlled fluid such as water, may be flowed out of the third tube of the three-tube exchanger 330. A second exchanger manifold seal 336 provides a fluid tight seal between the second tube of the three-tube exchanger 328 and the second exchanger manifold 332. A gas manifold 339 connects gas port 338 to the second tube of the three-tube exchanger 328, allowing for the introduction of gas, e.g., 0₂ and/or C0₂, into the second tube of the three-tube exchanger 328. An input valve 341 and media input port 342 facilitate the introduction of media into the first tube of the three-tube exchanger 326, and an injection port 340 allows for the injection of any suitable agent, e.g., one or more labeled substrates, into the media.

Bioreactor Perfusion System Including Fret Bioreactor Construct

[0048] With reference to FIGS. 12 - 15, another embodiment of a bioreactor

perfusion system according to the present disclosure is provided. This embodiment may be either a closed loop bioreactor perfusion system 100 or an open loop bioreactor perfusion system 200 as described above. Bioreactor perfusion system 500 includes a fret bioreactor construct 600 positioned in a cell and/or culture tube 304. A media delivery tube 344 extends through the fret bioreactor construct 600 and is configured to deliver media received from a concentric exchanger (106 or 206) through the fret bioreactor construct 600. In bioreactor perfusion system 300, concentric exchanger (106 or 206) is a three-tube exchanger, including a first tube of the three-tube exchanger 326, a second tube of the three-tube exchanger 328, and a third tube of the three-tube exchanger 330. Bioreactor perfusion system 300 includes a tube cap 306 which engages cell and/or culture tube 304. An outlet tube 312 and outlet connector 314 are configured to allow a medium, which has flowed through fret bioreactor construct 600 to exit the cell and/or culture tube 304. An inlet tube seal 308 positioned at the top of tube cap 306 engages inlet tube 310 providing a fluid-tight seal. In some embodiments, media delivery tube 344 and inlet tube 310 are two portions of the same tube structure. In other embodiments, they may be distinct structures.

[0049] Inlet tube 310 connects to the first tube of the three-tube exchanger 326

through inlet connector 316, and first exchanger manifold connector 322. A second tube connector seal 346 engages the second tube of the three-tube exchanger 328, providing a fluid tight seal. The second tube of the three-tube exchanger 328 extends through first exchanger manifold 318, a first exchanger manifold seal 320 providing a fluid-tight seal there between. First exchanger manifold inlet/outlet tube 324 provides a means by which a fluid, e.g., a temperature controlled fluid such as water, may be flowed into a third tube of the three-tube exchanger 330. The third tube of the three-tube exchanger 330 engages a tube connecting flange 352 of the first exchanger manifold 318.

[0050] At the opposite end of concentric exchanger (106 or 206) the third tube of the three-tube exchanger 330 engages a tube connecting flange 352 of second exchanger manifold 332. The first tube of the three-tube exchanger 326 and the second tube of the three-tube exchanger 328 extend through second exchanger manifold 332. A second exchanger manifold inlet/outlet tube 334 provides a means by which a fluid, e.g., a temperature controlled fluid such as water, may be flowed out of the third tube of the three-tube exchanger 330. A second exchanger manifold seal 336 provides a fluid tight seal between the second tube of the three-tube exchanger 328 and the second exchanger manifold 332. A gas manifold 339 connects gas port 338 to the second tube of the three-tube exchanger 328, allowing for the introduction of gas, e.g., 0₂ and/or C0₂, into the second tube of the three-tube exchanger 328. An input valve 341 and media input port 342 facilitate the introduction of media into the first tube of the three-tube exchanger 326, and an injection port 340 allows for the injection of any suitable agent, e.g., one or more labeled substrates, into the media.

Bioreactors

[0051] As described previously herein, the disclosed bioreactor perfusion systems may include a bioreactor in which cells and/or tissue are grown and/or monitored. A variety of bioreactors are known in the art, and in some embodiments of the present disclosure one or more of the concentric exchangers, and/or exchanger manifolds disclosed herein may be configured to operate in conjunction with one or more previously known bioreactors. In other embodiments, the present disclosure provides bioreactors and related bioreactor constructs which are specifically adapted to work with the bioreactor perfusion system components disclosed herein.

[0052] The bioreactors provided by the present disclosure include one or more

bioreactor constructs positioned in a cell/and or tissue culture tube, e.g., an NMR tube. The bioreactor constructs, when positioned in the cell/and or tissue culture tube, in conjunction with the walls of the cell and/or tissue culture tube, define a bioreactor chamber, e.g., a media-perfused space which facilitates the observation, analysis, growth and/or proliferation of cells and/or tissues (see, e.g., FIGs. 3-6 and 12-15). In general terms, a bioreactor construct according to the present disclosure is a solid construct that allows for the flow of media fluid into and out of the media-perfused space of the bioreactor but restricts the movement of cells and/or tissues out of the media-perfused space.

[0053] A bioreactor construct according to the present disclosure may be made from any material suitable for the proposed application of the bioreactor. For example, in some embodiments, a bioreactor construct according to the present disclosure includes or is prepared from a UV-cured polymer material. Where the application of the bioreactor is analysis via magnetic resonance spectroscopy, a suitable material may be one which is magnetic resonance (MR) compatible. For example, in some embodiments, a suitable material is a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water, e.g., a material having a magnetic susceptibility within 5% or 1% of the magnetic susceptibility of water.

[0054] A bioreactor construct according to the present disclosure may include or be prepared from a material exhibiting one or more of the following characteristics: MR matched magnetic susceptibility, biocompatibility, compatibility with one or more bio-solvents, water impermeability, suitable mechanical properties (e.g., tensile strength of about 35 MPa, elongation at break of about 2 to 3%, modulus of elasticity of about 4890 MPa, and hardness of about 93 Shore), suitable thermal properties (e.g., glass transmission temperature of about 42 °C), etc. A suitable material may be a curable material, e.g., a curable resin. In some embodiments, a suitable curable resin is a nanoparticle-filled light-curable resin, e.g., RC31 (NanoCure) resin available from EnvisionTEC, Germany.

[0055] A bioreactor construct according to the present disclosure may be sized and shaped to fit within a cell and/or tissue culture tube, e.g., an NMR tube. For example, in some embodiments a bioreactor construct according to the present disclosure is sized and shaped to fit within an approximately 10 mm outer diameter cell and/or tissue culture tube, e.g., an approximately 9 mm outer diameter cell and/or tissue culture tube, an approximately 8 mm outer diameter cell and/or tissue culture tube, an approximately 7 mm outer diameter cell and/or tissue culture tube, an approximately

6 mm outer diameter cell and/or tissue culture tube, an approximately 5 mm outer diameter cell and/or tissue culture tube, or smaller. In some embodiments, a bioreactor construct according to the present disclosure is sized and shaped to fit within an approximately 9 mm inner diameter cell and/or tissue culture tube, e.g., an approximately 8 mm inner diameter cell and/or tissue culture tube, an approximately

7 mm outer diameter cell and/or tissue culture tube, an approximately 6 mm outer diameter cell and/or tissue culture tube, an approximately 5 mm outer diameter cell and/or tissue culture tube, an approximately 4 mm inner diameter cell and/or tissue culture tube, or smaller.

[0056] In some embodiments, a bioreactor construct according to the present

disclosure has a diameter of about 10 mm or less, e.g., about 9 mm, about 8 mm, about 7 mm, about 6 mm, about 5 mm, about 4 mm, or less.

[0057] In some embodiments, a bioreactor construct according to the present

disclosure includes a first end and a second end and defines a central bore extending from the first end to the second end, wherein the central bore is configured to fluidically receive a media input from a first tube of a three-tube exchanger as described herein. In some embodiments, the central bore has a diameter of from about 0.5 mm to about 2 mm, e.g., from about 0.5 mm to about 1 mm.

[0058] A bioreactor construct according to the present disclosure may be prepared by any of a variety of suitable techniques, e.g., casting, injection molding, extrusion, 3- D printing, etc.

[0059] As discussed previously herein, a bioreactor construct according to the

present disclosure may be positioned in a cell/and or tissue culture tube, e.g., an MR (magnetic resonance) compatible tube. The bioreactor construct may define a media perfused space in the MR compatible tube through which media fluid may flow, but which is capable of retaining within it a biological material of interest. Biological materials of interest include, but are not limited to, particles, cells, tissues, and the like. In some embodiments, the biological material is suspended in fluid inside the bioreactor. In some embodiments, the biological material is immobilized on the bioreactor construct and/or the walls of the tube.

[0060] In some embodiments, as discussed herein, a cell/and or tissue culture tube of a disclosed bioreactor perfusion system includes a media delivery tube 344 (see FIGs. 4-6, and 13-15) positioned concentrically within the bore of a cell/and or tissue culture tube 304 which extends into/and or through a bioreactor construct according to the present disclosure. In some embodiments, the media delivery tube extends through a central bore of the bioreactor construct to the bottom of the cell/and or tissue culture tube 304. The media delivery tube may be used to deliver media fluid into the media perfused space of the bioreactor. In some embodiments, the media fluid then flows through the bioreactor construct and up the cell/and or tissue culture tube to an outlet tube 312. In this way, a controlled flow of media fluid may be supplied to the bioreactor.

Spindle Bioreactor Construct

[0061] As discussed previously herein, a bioreactor perfusion system according to the present disclosure may include a spindle bioreactor construct, e.g., a spindle bioreactor construct 400 positioned within a cell and/or tissue culture tube 304 in a bioreactor perfusion system 300. The spindle bioreactor construct 400 may find use, for example, in the growth and/or perfusion of tissues which can be wrapped, e.g., spirally wrapped, around a central shaft (or spindle) 404 of the spindle bioreactor construct 400.

[0062] Embodiments of a spindle bioreactor construct according to the present

disclosure are now described in greater detail with reference to FIGs. 7-11. In some embodiments, the spindle bioreactor construct 400 includes a central shaft 404 which defines a central bore 402 into or through which a media delivery tube 344 may be inserted to provide for perfusion of media into the bioreactor. The central shaft 404 may be porous or non-porous, or may include a porous or non-porous material. Where the central shaft is porous or includes a porous material, media may flow through the pores of the central shaft and into the media-perfused space of the bioreactor. The spindle bioreactor construct 400 may also include two ends, i.e., a first end 410 and a second end 412, which are connected by the central shaft 404. The spindle bioreactor construct 400 may also include one or more additional structures located around the central bore, such as one or more sidewalls 406 connected to the central bore via one or more intervening struts 408. In some embodiments, the spindle bioreactor construct 400 includes a plurality of sidewalls 406 extending the length of the spindle bioreactor construct 400 and defining sidewall openings 418 in the spindle bioreactor construct 400. In some embodiments, the spindle bioreactor construct includes a plurality of struts 408 extending perpendicularly from the central shaft 404 and connecting the central shaft 404 with the plurality of sidewalls 406.

[0063] In some embodiments, the first end 410 and the second end 412 of the spindle bioreactor construct 400 each define a plurality of openings or through holes 416 which, when the spindle bioreactor construct 400 is positioned in a cell and/or tissue culture tube 304, provide a flow path between the first end 410, the sidewall openings 418 and the second end 412.

[0064] The dimensions of the spindle bioreactor construct 400 may be selected and or modified depending on a variety of factors, including but not limited to, the dimensions of the cell and/or tissue culture tube 304, compatibility with the Bo field of an MR spectrometer, the biological material to be contained in the bioreactor, etc.

[0065] The outer diameter of the spindle bioreactor construct 400 may be selected depending on the inner diameter of the cell and/or tissue culture tube 304, e.g., a 5mm NMR tube, into which it is to be inserted and/or positioned. In some embodiments, the spindle bioreactor construct 400 has one or more dimensions as depicted in FIG. 33.

[0066] In some embodiments, the diameter of the central bore 402 is greater at the first end 410 of the spindle bioreactor construct 400 and the second end 412 of the spindle bioreactor construct 400 than at an intervening point along the central bore 402 between the first end 410 and the second end 412.

[0067] In some embodiments, the first end 410 and the second end 412 of the spindle bioreactor construct 400 each include a plurality of ridges 414 extending at least partially along the length of the bioreactor. In some cases, one or more of the plurality of ridges 414 may contact or slidably engage an inner wall of the cell and/or tissue culture tube 304 when inserted and/or positioned therein. The ridges 414 may project longitudinally from the first and/or second end (410, 412) with any suitable width and length, and may be located at any suitable location around the

circumference of the first and/or second end (410, 412). In some embodiments, each first and/or second end (410, 412) may include 4, 8, 10, 12, 14, 16 or more ridges equally spaced longitudinally.

[0068] In some embodiments, the spindle bioreactor construct 400 has a length of about 20 mm to about 50 mm, e.g., 30 mm to about 40 mm. In some embodiments, the spindle bioreactor construct 400 has a length of about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm or about 50 mm. In some embodiments, the spindle has an outer diameter compatible with insertion into a 5 mm NMR tube.

[0069] In an alternative embodiment, in addition to, or as an alternative to, a central shaft 404 which defines a central bore 402 into or through which a media delivery tube 344 may be inserted to provide for perfusion of media into the bioreactor, a bioreactor construct suitable for the growth of cells and/or tissues may include one or more horizontal cross-pieces which connect sidewalls 406. These cross-pieces may be spaced, e.g., equidistantly, along the length of the bioreactor construct, e.g., providing a vertical ladder-like structure along one or more sides of the bioreactor construct. The horizontal cross-pieces may have any suitable structure. For example, where the sidewalls 406 have a curved structure, the horizontal cross-pieces may have a curved structure with a curvature substantially similar to that of the sidewalls 406. Additional structures are also possible and considered within the scope of the present disclosure.

[0070] In some embodiments, corresponding horizontal cross-pieces, e.g., cross- pieces positioned across from each other at approximately the same level in the bioreactor construct, may be connected by one or more additional cross-pieces, e.g., providing a horizontal ladder-like structure relative to the length of the bioreactor construct. These cross-pieces provide a substrate and/or scaffold on which (or by which) cells, e.g., encapsulated cells and/or tissues may be retained and/or grown, e.g., in a layered, horizontal configuration. Such an embodiment may also include one or more through-holes or ducts in one or more sidewalls 406, which may facilitate media flow and perfusion of cells and/or tissues in the vicinity of the cross- pieces. For example, such through-holes or ducts may be positioned in sidewalls 406 in-between the "rungs" of the vertical ladder-like structure. While specific embodiments of the bioreactor constructs are provided herein, such embodiments are not intended to be limiting, and one of ordinary skill in the art would understand that a variety of modifications to the above structure are possible while falling within the scope of the present disclosure. One example of the above structure is depicted generally in FIG. 42.

Fret Bioreactor Construct

[0071] As discussed previously herein, a bioreactor perfusion system according to the present disclosure may include a fret bioreactor construct (baffle), e.g., a fret bioreactor construct 600 positioned within a cell and/or tissue culture tube 304 in a bioreactor perfusion system 500. The fret bioreactor construct 600 may find use, for example, in the growth and/or perfusion of suspended encapsulates, e.g.,

encapsulated cells, such as alginate-encapsulated cells, which can be suspended in the media-perfused space of a bioreactor utilizing a bioreactor construct 600.

[0072] Embodiments of a fret bioreactor construct according to the present

disclosure are now described in greater detail with reference to FIGs. 16-17. In some embodiments, a fret bioreactor construct 600 includes a central bore 602 extending from a first end 606 to a second end 608, and into or through which a media delivery tube 344 may be inserted to provide for perfusion of media into the bioreactor.

[0073] In some embodiments, the fret bioreactor construct 600 defines channels, through holes, pores, or the like, positioned external to the central bore 602 and has an overall shape and size suitable for insertion and/or positioning in a cell and/or tissue culture tube, e.g., a cell and/or tissue culture tube 304, such that when inserted and/or positioned, the fret bioreactor construct 600 acts to restrict the movement of a biological material, while allowing media fluid to pass. In some embodiments, the through holes are positioned radially around the central bore, and in some embodiments, the through holes extend parallel to the central bore.

[0074] In some embodiments, the fret bioreactor construct 600 defines a plurality of through holes 604 that run parallel to the central bore 602 and extend from first end 606 to second end 608. The diameter of the through holes 604 may be selected so as to provide for a desired flow of media fluid, while preventing the passage of biological materials of interest, such as cells and/or tissue, through the fret bioreactor construct 600 and out of the media-perfused space of the bioreactor.

[0075] The dimensions of the fret bioreactor construct 600 may be selected

depending on a variety of factors, including but not limited to, the dimensions of the cell/and or tissue culture tube 304, compatibility with the Bo field of an MR spectrometer, the biological material to be contained in the bioreactor, etc. The outer diameter of the fret bioreactor construct 600 may be selected depending on the inner diameter of the cell and/or tissue culture tube 304, e.g., a 5 mm NMR tube, into which it is to be inserted and/or positioned. In some embodiments, the fret bioreactor construct 600 has one or more dimensions as depicted in FIG. 32.

[0076] In some embodiments, the central bore 602 has a diameter of about 1 mm to about 2 mm, e.g., about 1.2 mm to about 1.8 mm, about 1.4 mm to about 1.6 mm, or about 1.5 mm. In some embodiments, the through holes 604 have a diameter of from about 0.2 to about 0.6 mm. In certain embodiments, the through holes have a diameter of about 0.4 mm.

[0077] In some embodiments, the through holes 604 of the fret bioreactor construct

600 have a diameter of about 500 μιη or less, such as about 400 μιη or less, about 350 μιη or less, about 300 μιη or less, about 250 μιη or less, about 200 μιη or less, about 150 μιη or less, or about 100 μιη or less. In some embodiments, the plurality of through holes 604 in the fret bioreactor construct 600 have a mean diameter of about 500 μιη, about 400 μιη, about 300 μιη, about 250 μιη, about 200 μιη, about 150 μιη, or about 100 μιη. In some embodiments, the through holes 604 have a diameter of about 300 μιη.

[0078] It should be noted that the diameter of the through holes may be configured based on the size of the encapsulates the fret bioreactor construct is configured to retain and perfuse. Accordingly, the above dimensions are merely exemplary, and through holes 604 having diameters greater and smaller than those described above are contemplated and considered within scope of the present disclosure. For example, in some embodiments, the through holes 604 are sized to have a diameter which is about 90% of the average diameter (or greatest dimension) of the encapsulates, e.g., about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of the average diameter (or greatest dimension) of the encapsulates. In some embodiments, the fret bioreactor construct 600 includes 10 or more through holes 604, such as 15 or more, 20 or more, 25 or more, 30 or more, 50 or more, or 100 or more through holes 604.

[0079] In some embodiments, the fret bioreactor construct 600 is porous. In some embodiments, the fret bioreactor construct 600 is composed of a material that is inherently porous. In some embodiments, the through holes 604 of the fret bioreactor construct 600 are introduced into a nonporous material. [0080] In some embodiments, the length of the fret bioreactor construct 600 from the first end 606 to the second end 608 is from about 2 mm to about 6 mm, e.g., from about 3 mm to about 5 mm, or about 4 mm.

Tube Cap

[0081] As discussed previously herein, the bioreactor perfusion systems according to the present disclosure may include a tube cap, e.g., a tube cap 306, configured to engage a cell and/or tissue culture tube, e.g., a cell and/or tissue culture tube 304, and facilitate media delivery to a bioreactor and media exit from a cell and/or tissue culture tube.

[0082] In some embodiments, the tube cap includes a body defining an inlet port, an outlet port, a tube-engagement opening, and an internal chamber connecting the inlet port, the outlet port and the tube engagement opening. In some embodiments, the body of the tube cap includes an internal wall at least partially defining the internal chamber, wherein the internal wall defines a channel configured to facilitate liquid flow through the internal chamber and out the outlet port. In some embodiments, the channel has a spiral structure.

[0083] Embodiments of a tube cap according to the present disclosure are now

described in greater detail with reference to FIGs. 18-21. In some embodiments, a tube cap 306 defines a tube engagement opening 358 configured to engage the opening of a cell and/or tissue culture tube 304. Tube cap 306 may also define a tube inlet port 368 configured to receive media delivery tube 344, an inlet tube seal receiving area 366 configured to receive inlet tube seal 308, internal threads 362 for receiving external threads present on a cell and/or tissue culture tube, e.g., an NMR tube, and a media outlet port 364 through which media fluid may pass out of the cell and/or tissue culture tube 304.

[0084] Any suitable sealing means may be utilized in connecting the tube cap 306 to the cell and/or tissue culture tube 304. In some embodiments, the tube cap 306 includes one or more O-rings positioned in the internal chamber so as to engage an external wall of a cell and/or tissue culture tube 304. In some embodiments, an O- ring seal 360 may be utilized to provide a fluid-tight seal between an outer wall of the cell and/or tissue culture tube 304 and the tube cap 306. In some embodiments, the cell and/or tissue culture tube 304 and the tube cap 306 are threaded to facilitate a threaded engagement. [0085] In some embodiments, a tube cap according to the present disclosure, e.g., a tube cap 306, includes one or more connectors which, individually or together, provide a fluid flow path from the first tube of a three-tube exchanger 326 through a tube inlet port 368 of the tube cap 306 to the bioreactor.

[0086] In some embodiments, the tube cap 306 is connected to an outlet tube 312 and an outlet connector 314 which provides for the flow of media fluid out of the tube. The tube cap 306 may be further connected to an inlet tube 310 via an inlet tube seal 308. The inlet tube 310 may be fluidically connected to the media delivery tube 344. Alternatively, inlet tube 310 and media deliver tube 344 may be two parts of the same tube structure. In some embodiments, the media outlet port 364 of the tube cap receives perfusion media after it has passed through the bioreactor. The outlet tube 312 is fluidically connected to the cell and/or culture tube 304. In some

embodiments, a perfusion media reservoir is fluidically connected to the outlet tube 312. The tube cap 306 may include a tube inlet port 368 for connection to the inlet tube 310 via the inlet tube seal 308 and a tube engagement opening 358 with or without an O-ring seal 360 for connection to the cell/and or tissue culture tube 304. In some embodiments, the tube engagement opening 358 includes a threaded portion for attaching via threaded engagement to a threaded cell/and or tissue culture tube 304.

[0087] In some embodiments, the tube cap 306 further provides for connection of the inlet tube 310 to a first exchanger manifold 318 via an inlet connector 316, a first exchanger manifold connector 322, and a second tube connector seal 346.

[0088] A tube cap according to the present disclosure, e.g., a tube cap 306, may be made from any material suitable for the proposed application of the bioreactor perfusion system. For example, in some embodiments, a tube cap according to the present disclosure includes or is prepared from a UV-cured polymer material. Where the application of the bioreactor perfusion system is analysis via magnetic resonance spectroscopy, a suitable material may be one which is magnetic resonance (MR) compatible. For example, in some embodiments, a suitable material is a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water, e.g., a material having a magnetic susceptibility within 5% or 1% of the magnetic susceptibility of water.

[0089] A tube cap according to the present disclosure may include or be prepared from a material exhibiting one or more of the following characteristics: MR matched magnetic susceptibility, biocompatibility, compatibility with one or more bio- solvents, water impermeability, suitable mechanical properties (e.g., tensile strength of about 35 MPa, elongation at break of about 2 to 3%, modulus of elasticity of about 4890 MPa, and hardness of about 93 Shore), suitable thermal properties (e.g., glass transmission temperature of about 42 °C), etc. A suitable material may be a curable material, e.g., a curable resin. In some embodiments, a suitable curable resin is a nanoparticle-filled light-curable resin, e.g., RC31 (NanoCure) resin available from EnvisionTEC, Germany.

[0090] A tube cap according to the present disclosure may be prepared by any of a variety of suitable techniques, e.g., casting, injection molding, extrusion, 3-D

printing, etc.

Concentric Exchanger

[0091] As discussed previously herein, the bioreactor perfusion systems according to the present disclosure may include an exchanger to facilitate equilibration of media fluid prior to entry into a bioreactor. A variety of exchangers are known in the art, and in some embodiments of the present disclosure one or more of the bioreactors or related bioreactor constructs disclosed herein may be configured to operate in conjunction with one or more previously known exchangers. In other embodiments, the present disclosure provides exchangers which are specifically adapted to work with the bioreactor perfusion system components disclosed herein.

[0092] Exchangers according to the present disclosure are now described in greater detail with reference to FIGs. 1- 3, 12 and 25-28. In some embodiments, the present disclosure provides a concentric exchanger, e.g., a concentric exchanger 106 or 206. The concentric exchanger provides for the supply of an equilibrated media fluid to the bioreactor, where the temperature and/or gas content of the media fluid may be adjusted before the fluid enters the bioreactor. In addition, an amount of one or additional components may be added to the media fluid via an inlet port or injection port in fluid communication with the concentric exchanger at any of a variety of suitable positions within the bioreactor perfusion system.

[0093] In some embodiments, a concentric exchanger 106 or 206 according to the present disclosure includes a first elongate structure defining a first bore extending lengthwise therethrough, a second elongate structure defining a second bore extending lengthwise therethrough, and a third elongate structure defining a third bore extending lengthwise therethrough, wherein the first elongate structure is positioned concentrically within the second bore of the second elongate structure and the second elongate structure is positioned concentrically within the third bore of the third elongate structure. The elongate structures may have any suitable cross- sectional shape, e.g., round, oval, polygonal, etc. In some embodiments, as discussed in greater detail below, the elongate structures are tubular structures.

[0094] In some embodiments, the concentric exchanger 106 or 206 is a three-tube exchanger as depicted in FIGs. 3, 12 and 25-28, where the three-tube exchanger includes a first tube 326, a second tube 328 and a third tube 330. The first tube 326 may be positioned concentrically within the bore of the second tube 328, and the second tube 328 may be positioned concentrically within the bore of the third tube 330. As such, the arrangement of inner and outer surfaces of the first, second and third tubes of the three-tube exchanger may define three separate flow paths through which liquids and/or gases may flow. In some embodiments, the three elongate structures of the concentric exchanger, e.g., the three tubes of a three-tube exchanger, provide for the flow of a temperature-controlled fluid (e.g., water), one or more gases at a desired pressure and a media fluid. One or more of the tubes, e.g., the outer third tube 330, may be insulated to minimize heat loss. In some cases, the third tube 330 includes an external insulation layer. One or more of the tubes may be gas permeable to allow a gas to permeate from the bore of one elongate structure to another or gas impermeable to prevent such permeation.

[0095] As used herein, the term "gas permeable", "relatively gas permeable" and the like refer to a material having an 0₂ and/or C0₂ permeability at 25 °C which is equal to, within 20% of, or greater than the 0₂ and/or C0₂ permeability of silicone at 25°C.

[0096] As used herein, the terms "gas impermeable", "relatively gas impermeable" and the like refer to a material having an 0₂ and/or C0₂ permeability at 25 °C which is equal to, within 20% of, or less than the 0₂ and/or C0₂ permeability of fluorinated ethylene propylene (FEP) at 25 °C.

[0097] In some embodiments, the concentric exchanger 106 or 206 is configured to accept a fluid media in first tube 326, a gas for equilibration of the media in second tube 328, and a temperature-controlled fluid for regulating temperature of the media in third tube 330.

[0098] In some embodiments, the temperature-controlled fluid flows through the space between the third tube 330 and the second tube 328. As such, the third tube 330 may be fluidically coupled to a temperature-controlled fluid flow. In certain embodiments, the third tube 330 includes an external insulation layer. In some embodiments, the temperature-controlled fluid flow is a temperature-controlled water flow.

[0099] In some embodiments, the space between the second tube 328 and the first tube 326 includes a gas at a desired pressure. As such, the second tube 328 may be fluidically coupled to a pressurized gas input. In some embodiments, the walls of the first tube 326 are gas permeable such that gas in the bore of the second tube 328 may pass into the bore of first tube 326. In some embodiments, the first tube 326 is gas permeable, and the second tube 328 is gas impermeable such that gas under pressure (e.g., air, air/C0₂, C0₂ or 0₂) can be forced to dissolve into the media fluid flowing in the first tube 326.

[00100] In some embodiments, the first tube 326 may be fluidically coupled to a perfusion media input. As such, in some embodiments, the fluid media flows inside the bore of first tube 326.

[00101] Any suitable dimensions for the tubes of the concentric exchanger, e.g., a three-tube exchanger, may be selected, including dimensions such as inner (ID) and outer tube diameters (OD), tube wall thickness and tube length. For example, diameter of the three-tube exchanger may be selected so that the exchanger is compatible with the diameter of a cell/and or tissue culture tube, e.g., a cell and/or tissue culture tube 304, to which it may be connected. In some embodiments, the concentric exchanger has an outer diameter compatible with a standard NMR bore diameter (e.g., ID 50 mm).

[00102] The concentric exchanger may have any suitable length. For example, the length of the exchanger may be selected, in conjunction with the flow rate, to provide for equilibration of the temperature of a fluid, and/or equilibration of a gas content of the fluid, by the time the fluid exits the exchanger. The length of the exchanger may be selected according to a desired flow rate of a fluid, and/or the desired temperature and/or pressure of gas in one or more of the tubes.

[00103] In some embodiments, the third tube 330 of the 3-tube exchanger has an outer diameter (OD) of from about 10 mm to about 15 mm, an inner diameter (ID) of from about 8 mm to about 12 mm, and a thickness of about 3 mm or more. In some embodiments, the third tube 330 has an OD of about 12.7 mm and an inner diameter of about 9.5 mm. [00104] In some embodiments, the second tube 328 has an OD of from about 4 mm to about 9 mm, an ID of from about 3 mm to about 7 mm, and a thickness of about 1 mm or more. In some embodiments, the second tube 328 has an OD of about 6.4 mm (e.g., 6.35 mm) and an ID of about 4.8 mm (e.g., 4.76 mm).

[00105] In some embodiments, the first tube 326 has an OD in the range from about

0.7 mm to about 1.1 mm, an ID in the range from about 0.3 mm to about 0.7 mm, and a thickness of about 0.4 mm or more. In some embodiments, the first tube 326 has an OD of about 0.9 mm (e.g., 0.94 mm) and an ID of about 0.5 mm (e.g., 0.51 mm).

[00106] In some embodiments, the first tube 326 has an OD of about 0.037" and an ID of about 0.02". In some embodiments, the second tube 328 has an OD of about 1/4" and an ID of about 3/16". In some embodiments, the third tube 330 has an OD of about 1/2" and an ID of about 3/8".

[00107] The concentric exchanger may be comprised of any suitable materials

consistent with their proposed use. Generally, such materials will be relatively flexible and leak-tight or otherwise liquid impermeable. Suitable materials may include, but are not limited to, plastic, polyethylene, polypropylene, rubber, Tygon, fluorinated ethylene propylene (FEP), polytetrafluoroethylene (PTFE), and a perfluoroalkoxy (PFA), silicone, silastic, and the like. Suitable materials may be either transparent or opaque. For first tube 326 of the three-tube exchanger, a suitable material may be one which is relatively gas permeable and has low chemical reactivity, such as Silastic™ (silicone) tubing. For the second tube 328 of the three- tube exchanger, a suitable material may be one which is relatively impermeable to gas, e.g., 0₂ and C0₂, and has relatively high heat transfer conductivity, e.g., fluorinated ethylene propylene (FEP). For the third tube 330 of the three-tube exchanger, a suitable material may be one which is relatively insulative.

Alternatively, an insulative layer may be added to third tube 330 to retain heat within the bore of the tube.

[00108] In some embodiments, the first tube 326 of the three-tube exchanger is

composed of silicone. In some embodiments, the second tube 328 of the three-tube exchanger is composed of FEP. In some embodiments, the third tube 330 of the three-tube exchanger is composed of Tygon. Any suitable type of Tygon tubing may be utilized. Exchanger Manifolds

[00109] The concentric exchanger of the present disclosure may be connected to an exchanger manifold at either or both ends. Any suitable manifolds, and

configurations thereof, may be utilized in the subject bioreactor-perfusion systems to introduce or remove fluids and gases of interest from the exchanger and/or bioreactor. As used herein, the term "manifold" refers to a branched tube or branched connector through which fluids and/or gases may pass. Any suitable fittings, joints, connectors, ports, flanges, seals, tubes, and the like, may be utilized in connecting the components of the system (e.g., manifolds, tubes, connectors, seals, ports, etc.) in configuring the bioreactor perfusion systems.

[00110] In some embodiments, a first exchanger manifold is coupled to the concentric exchanger at a first end of the concentric exchanger, the first exchanger manifold including a body defining an internal chamber and first, second and third ports connected via the internal chamber. The first port of the first exchanger manifold interfaces with the third bore of the third elongate structure of the concentric exchanger, and the first and second elongate structures of the concentric exchanger extend through the first and second ports of the first exchanger manifold. The first exchanger manifold provides a flow path between the third bore of the third elongate structure of the concentric exchanger and the third port of the first exchanger manifold. In some embodiments, the first port of the first exchanger manifold engages the third bore of the third elongate structure of the concentric exchanger via press-fit engagement. Alternatively, or in addition, one or more adhesives or glues may be applied to seal the connection between the first port of the first exchanger manifold and the third bore of the third elongate structure. In some cases, the first port of the first exchanger manifold includes a tube-connecting flange including external barbs or ridges to effect the press-fit engagement. In some embodiments, a seal is positioned in the second port of the first exchanger manifold between the body of the exchanger manifold and the second elongate structure of the concentric exchanger.

[00111] In some embodiments, a second exchanger manifold is coupled to the

concentric exchanger at a second end of the concentric exchanger, the second exchanger manifold including a body defining an internal chamber and first, second and third ports connected via the internal chamber. The first port of the second exchanger manifold interfaces with the third bore of the third elongate structure of the concentric exchanger, and the first and second elongate structures of the concentric exchanger extend through the first and second ports of the second exchanger manifold. The second exchanger manifold provides a flow path between the third bore of the third elongate structure of the concentric exchanger and the third port of the second exchanger manifold. In some embodiments, the first port of the second exchanger manifold engages the third bore of the third elongate structure of the concentric exchanger via press-fit engagement. Alternatively, or in addition, one or more adhesives or glues may be applied to seal the connection between the first port of the second exchanger manifold and the third bore of the third elongate structure. In some cases, the first port of the second exchanger manifold includes a tube-connecting flange including external barbs or ridges to effect the press-fit engagement. In some embodiments, a seal is positioned in the second port of the second exchanger manifold between the body of the exchanger manifold and the second elongate structure of the concentric exchanger.

[00112] Embodiments of the exchanger manifolds of the present disclosure and their relationships to other components of the bioreactor perfusion systems of the present disclosure are now described in greater detail with reference to FIGs. 1-3, 12 and 25- 28. A concentric exchanger (106, 206) according to the present disclosure, e.g., a three-tube exchanger, may be connected to a first exchanger manifold (104, 204, 318) and a second exchanger manifold (108, 208, 332). The exchanger manifolds may provide for the maintenance of gas pressure and the flow of fluids into and/or out of one or more elongate structures, e.g., tubes, of the concentric exchanger. In some embodiments, first and second exchanger manifolds 318 and 332 define exchanger manifold inlet/outlet ports 350, which in turn provide for connection to exchanger manifold inlet/outlet tubes 324 and 334, which allow a temperature- controlled fluid (e.g., water) to flow into and out of the third tube 330 of a three-tube concentric exchanger. It should be noted that in embodiments of the present disclosure, the temperature-controlled fluid may flow in either direction, i.e., into inlet/outlet tube 324 and out of inlet/outlet tube 334, or vice versa.

[00113] In some embodiments, the concentric exchanger e.g., a three-tube concentric exchanger, is connected to the first and/or second exchanger manifolds (318, 332) via a tube-connecting flange 352. In some embodiments, the first exchanger manifold 318 provides for the flow of media fluid through the exchanger manifold and into a bioreactor, optionally via one or more connectors and inlet tubes. In some embodiments, second exchanger manifold 332 provides for the flow of media fluid and gas of a desired pressure into the concentric exchanger.

[00114] A variety of inlet tubes, connectors, ports and/or seals may be utilized in configuring the concentric exchanger (106, 206) and exchanger manifolds (318 and 332) for operation. In some embodiments, the first exchanger manifold 318 includes one or more seals (320, 346) and connectors 322 to facilitate fluid connection of the concentric exchanger (106, 206) to a cell and/or tissue culture tube 304.

[00115] In some embodiments, the second exchanger manifold 332 fluidically

connects the third tube of the 3-tube exchanger 330 to an inlet/outlet tube 334 via an inlet/outlet port 350. The second exchanger manifold 332 may be further connected to a gas manifold 339 to fluidically connect the second tube of the 3-tube exchanger 328 to a gas port 338. In some embodiments, the bioreactor perfusion system includes a pressurized gas reservoir coupled to the second elongate structure (e.g., second tube 328) of a concentric exchanger. The second exchanger manifold 332 may be further connected, optionally via the gas manifold 339, to an input valve 341 which provides for fluidic connection of the first tube 326 or elongate structure of the three-tube exchanger to a media fluid (e.g., from a media reservoir) via media input port 342. Input valve 341 also provides an optional connection point for or an injection port 340, through which any suitable material or agent, e.g., a labeled substrate, may be injected into the media fluid.

[00116] In some embodiments, a bioreactor system according to the present disclosure includes an injection port coupled to the first elongate structure, e.g., first tube 326, of a concentric exchanger. In some cases, the injection port may find use in the addition of one or more hyperpolarized substrates into the bioreactor via the media fluid.

[00117] First and/or second exchanger manifolds according to the present disclosure, e.g., first and/or second exchanger manifolds 318 and 332, may be made from any material suitable for the proposed application of the bioreactor perfusion system. For example, in some embodiments, an exchanger manifold according to the present disclosure includes or is prepared from a UV-cured polymer material. Where the application of the bioreactor perfusion system is analysis via magnetic resonance spectroscopy, a suitable material may be one which is magnetic resonance (MR) compatible. For example, in some embodiments, a suitable material is a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water, e.g., a material having a magnetic susceptibility within 5% or 1% of the magnetic susceptibility of water.

[00118] The first and/or second exchanger manifolds may include or be prepared from a material exhibiting one or more of the following characteristics: MR matched magnetic susceptibility, biocompatibility, compatibility with one or more bio- solvents, water impermeability, suitable mechanical properties (e.g., tensile strength of about 35 MPa, elongation at break of about 2 to 3%, modulus of elasticity of about 4890 MPa, and hardness of about 93 Shore), suitable thermal properties (e.g., glass transmission temperature of about 42 °C), etc. A suitable material may be a curable material, e.g., a curable resin. In some embodiments, a suitable curable resin is a nanoparticle-filled light-curable resin, e.g., RC31 (NanoCure) resin available from EnvisionTEC, Germany.

[00119] Exchanger manifolds according to the present disclosure may be prepared by any of a variety of suitable techniques, e.g., casting, injection molding, extrusion, 3- D printing, etc.

Additional System Components and Features

[00120] In some embodiments, a bioreactor perfusion system according to the present disclosure provides for recirculation of one or more fluids (e.g., a temperature- controlled fluid or media) from an outlet downstream of the bioreactor back to an inlet or a fluid reservoir upstream of the bioreactor. In some embodiments, a perfusion media reservoir receives perfusion media after it has passed through the bioreactor. As such, the system may be referred to as a closed system. In some embodiments, the bioreactor perfusion system is open such that one or more fluids enter the system fresh from a reservoir, and are not recirculated after exiting the outlet.

[00121] Bioreactor perfusion systems according to the present disclosure may include a pump for circulating or flowing one or more fluids through the system, such as a peristaltic pump or a water bath pump. In some embodiments, a pump is configured to pump perfusion media from a perfusion media reservoir through the first bore of the first elongate structure of a concentric exchanger, e.g., a first tube 326 of a three- tube exchanger. In some embodiments, a temperature-controlled fluid (e.g., water) flow rate of about 0.5 mL/minute and a reservoir temperature of about 50°C may be used to provide a resulting media fluid at the outlet end of the exchanger having a temperature 37°C. In some embodiments, the pressure of gas (e.g., 95% air/5% C0₂, air, or 0₂) in the concentric exchanger is controlled to provide for media fluid at the outlet of the concentric exchanger having a desired concentration of one or more gases in the fluid. In some embodiments, the oxygen concentration in the media fluid at the outlet of the concentric exchanger is about 30%. The residence time of the media fluid in the concentric exchanger can also be controlled (e.g., by adjusting the flow rate and/or length of concentric exchanger) to provide for a desired temperature and gas concentration. The parameters of the concentric exchanger and the inputs thereto may be varied as needed to provide for a media fluid having any suitable temperature and gas concentration.

[00122] In some embodiments, the system includes one or more detectors and/or sensors for monitoring the characteristics of the media fluid, e.g., temperature, oxygen concentration, etc. Any suitable detectors and/or sensors may be utilized, including but not limited to, a fiber optic based oxygen sensor and a temperature sensor. In some embodiments, the one or more detectors and/or sensors may provide feedback to control the parameters of the exchanger configuration and/or inputs thereto, such as temperature of the heat exchanger fluid flowing into the exchanger, pressure of the gas at the gas port, and flow rates of the fluids.

EXAMPLES

[00123] 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 present invention, 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 accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., s or sec, second(s); min, minute(s); h or hr, hour(s); and the like. Hyperpolarized MR/PET compatible bioreactor for characterization of clinically translatable companion biomarkers

[00124] The goal of this study was to develop a 5mm MR-compatible platform

interfaced with hyperpolarized (HP) MR, for small cultures of immortal prostate cancer cells, and then extend these methods to positron emission tomography (PET) imaging to characterize synergistic probes for translation. This system was utilized to characterize biomarkers for response to a novel cancer therapeutic as well as metabolism in primary human prostate tissue cultures.

Materials and Methods

[00125] Cell Culture: Prostate cancer cells (PC-3) were cultured in T 150 cm flasks with DMEM medium (supplemented with 10% FBS and Penicillin/Streptomycin). Prostate tissue slices were prepared as follows: Fresh tissue cores (8-mm diameter) from radical prostatectomy specimens were embedded in agarose, mounted in a Krumdieck tissue slicer (Alabama Research and Development, Munford, AL) and rapidly sectioned (250-300um thickness) while immersed in chilled physiologic fluid. The tissue slices were then cultured in the same PFMR-4A medium as cell studies on a rotator inside of a standard cell culture incubator as described in Zhao et al. (2010) Tissue slice grafts: an in vivo model of human prostate androgen signaling. Am. J. Pathol. 177(l):229-239, the disclosure of which is incorporated by reference herein.

[00126] MR/PET compatible Bioreactor: For cell studies, 10 million PC-3 cells were suspended in 2% alginate and cross-linked in a 150mM CaCl₂ solution for encapsulation. For prostate TSCs, 4 slices were perfused in a custom-designed cartridge construct. Cells and primary tissue were cultured in a custom-designed 5mm MR-compatible bioreactor system. The system utilized a completely enclosed perfusion system, providing a continuous flow of 37°C medium (analogous to the culture medium) dynamically oxygenated with 95% Air/5% C0₂.

[00127] Oxygen Measurements: Oxygen measurements were made by interfacing a temperature calibrated fiber optic based 0₂ sensor (Ocean Optics) before and after the bioreactor chamber. These were used to determine the oxygenation level the exchanger achieved.

[00128] NMR Studies: All NMR data were acquired on a narrow-bore 11.7T Varian

13

INOVA (125MHz C) equipped with a 5mm broadband probe. Cell viability was

31 31

assessed acquiring P spectra (202MHz P) with a 90° pulse and acquire sequence 13

(nt=1024, at=ls, T_R=3s) to assess βΝΤΡ resonance. [1- C]pyruvate was

hyperpolarized using the Hypersense™ (Oxford Instruments) as described in Keshari KR, et al. (2010) Hyperpolarized (13)C spectroscopy and an NMR-compatible bioreactor system for the investigation of real-time cellular metabolism. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine 63(2):322-329, the disclosure of which is incorporated by reference herein. 1 mL of 4mM pyruvate was injected

13

into the bioreactor where C NMR spectra were acquired in intervals of 3 sees using 10° pulses for 300 sees. Peak integrals were calculated for each resonance and fluxes were calculated for label conversion to HP lactate.

[00129] MicroPET Studies: A small animal microPET/CT scanner (Inveon, Siemens

Medical Solutions, USA) was used for PET imaging. Medium containing 5 ^CilmL of 1¹8⁰F FDG was perfused through the bioreactor system for 40 min and washed out for 40 min. For the purposes of higher throughput, 4 bioreactors were run concurrently in the PET detector, one containing empty alginate encapsulates as a control. Post-washout, the reactor region was defined as a region of interest and activity was calculated relative to control.

[00130] Histopathology: After perfusion in the 5mm bioreactor, encapsulated cells were fixed in formalin and sectioned. These were stained for hematoxylin & eosin (for structure), KI-67 (for proliferation) and Caspase-3 (for apoptosis) using standard methods.

[00131] mRNA expression: Total RNA was isolated from frozen PC-3 cells (n=6) using the TriPure Isolation Reagent (Roche) and reverse transcribed using

Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. PCR primers were obtained from Applied Biosciences (Life

Technologies, CA, USA), and real-time PCR determination of cDNA amounts was performed. Relative expression to control gene Cyclophilin was determined using the ACt method. Primers were used for monocarboxylate transporters 1 and 4 (mctl,4), lactate dehydrogenase A and B (ldhA and B) as well as glucose like transporter 1 (glutl) and hexokinase 2 (hk2).

[00132] Lactate Dehydrogenase Activity: Lactate dehydrogenase (LDH) activity was measured spectrophotometrically by quantifying the linear decrease in NADH absorbance at varying pyruvate (sodium salt) concentrations at 339 nm for 10 minutes. Semi-confluent cells were harvested and 10 million cells were lysed (in buffer with 50 mM Tris pH 8.2, 2 mM DTT, 2 mM EDTA, 1% Triton x-100). LDH assays were then carried out using standard methods. LDH activity was plotted against the pyruvate concentration to arrive at the maximum velocity (Vmax) and the Michaelis-Menten constant (K_m) using the Lineweaver- Burke plot. Total protein measurement was carried out by the calorimetric Bradford reagent method (Quick Start Bradford Protein Assay from BioRad Laboratories, Hercules, CA, USA).

[00133] Data Analysis and Statistics: All NMR data was processed using a

combination of ACD Lab ID and 2D NMR processor (version 9; ACD/Labs, Toronto, Canada). Peak areas or volumes were integrated and used to derive the necessary concentrations. All statistics were calculated using JMP software (SAS Corporation, Cary, NC). All PET data was processed using open source AMIDE software. Significance was reported using a two-sided Student's t-test for all comparisons and a p-value<0.05.

Results

[00134] 5mm MR-compatible Bioreactor Design: In order to design an appropriate system to support living cultures within a high-resolution NMR, a number of design constraints were used. These constraints are not only governed by the needs of perfusion in real time, but also the ability to create the appropriate geometry to yield useable data. Firstly, the perfusion system was designed to not only provide a constant supply of nutrients and remove waste but also minimize this perfusion volume and adapt the apparatus for the NMR. A 3 concentric tube exchanger was designed (FIG. 30) to create the appropriate medium temperature and gas equilibration before the fluid enters the bioreactor (FIG. 29). The outermost tube (OD 12.7mm, ID 9.5mm) was made of Tygon and under continuous pumping via a circulating water bath, heated in order to create heat transfer inward. This tubing was insulated on the outside so as to minimize heat loss. The second inner tubing was composed of fluorinated ethylene propylene (FEP, OD 6.35mm, ID 4.76mm), under pressure at 5 PSI of carbogen (95% Air/ 5% C0₂). FEP tubing is relatively impermeable to gases and under pressure can force oxygen and C0₂ into the circulating medium as a function of their henry's law coefficients. The innermost tube, composed of silicone (OD 0.94mm, ID 0.51mm) contains inflow medium whose rate depends on the perfused reactor (Q = 0.1 - 1 mL/min). The exchanger overall diameter was configured such that it was compatible with a standard NMR narrow bore diameter (approximate ID 50mm). [00135] Custom manifolds were designed (FIG. 31) to interface the 3 tube exchanger for perfusion with both a peristaltic pump (medium) and water bath pump. The opposite side was designed for minimal volume connection to the top of the bioreactor construct (FIG. 31). Two bioreactor constructs were developed, one for perfusion of suspended encapsulates (FIG. 32) where a fret was implemented with 300 μιη diameter perfusion holes and a second spindle design for the perfusion of living primary tissues (FIG. 33). Additionally a custom cap was designed for medium delivery and recirculation on top of the NMR tube and interfaced to the exchanger (FIG. 34). Both construct arrangements were designed in CAD software

(Solidworks) and 3D printed using a UV Cured polymer (Perfactory 3,

EnvisionTEC). This allowed for the achievement of fine in plane resolution necessary to utilize these construct designs. While these parts are re-useable, they are easily replaced given the low-cost nature of their construction. Recirculation was achieved via a return FEP tubing to the medium reservoir (FIG. 29). This

configuration created the optimal condition for both modulation of oxygen concentration in medium as well as temperature. At a rate of 0.5 mL/min and water bath concentration of 50°C, the resulting medium at the end of the exchanger had an oxygen concentration of 30% and a temperature of 37°C with an estimated residence time of 24s.

[00136] High-resolution NMR generally requires the ability to homogenize the Bo field surrounding any flow cell in place. Given the reduction in volume and distribution of the sample in the Z direction, relative to larger reactor constructs (e.g. >10mm), a line-width of less than lHz in ^lH (at 11.7T) was easily achieved in perfusion medium alone through both the encapsulate and tissue spindle constructs, using a standard 24 element shim set. This is because the material utilized in the construct has a similar magnetic susceptibility to that of water. When encapsulates or tissue was introduced into the system, an average line-width of 10.9 ± 0.4 Hz was achieved (FIG. 35). This high mass sensitivity (filling factor) with ideal

homogeneity under perfusion was critical for the acquisition of multinuclear NMR data of small living cell cultures.

[00137] Cell viability with study in the MR-compatible bioreactor: In order to

determine the impact of continuous perfusion on living cell cultures in this bioreactor design, immortal prostate cancer (PC-3) cells were encapsulated and perfused for up to 48 hours. At each time point, encapsulates were histopathologically assayed for cell integrity (hematoxylin and eosin, H&E), changes in proliferation (Ki-67) and apoptosis (Caspase-3). Representative histologic sections demonstrate the preservation of cell architecture and health during the above perfusion period (FIG. 36). Encapsulated PC-3 cells continued to proliferate at an analogous rate with perfusion in the reactor (24 hr) with no significant change in apoptosis. These data support the preservation of cell homeostasis and growth within the bioreactor construct utilizing these flow conditions.

[00138] Steady State Bioenergetics of encapsulated PC-3 cells: Electrostatically

encapsulated PC-3 cells in alginate microspheres were then perfused within the 5mm MR-compatible bioreactor inside of a standard high-resolution NMR device.

31

Acquired P NMR spectra (FIG. 37, Panel B) demonstrate both the high quality data acquired as well as the ability to resolve and quantify resonances of interest, including the nucleotide triphosphates (NTPs, predominantly ATP), the

phosphomonoesters (PC - phosphocholine, PE - phosphoethanolamine) and phosphodiesters (GPC - glycerophosphocholine, GPE - glycerophosphoethanolmame). These resonances were quantified using an electronic reference standard (ERETIC) and scaled directly to encapsulated cell density (FIG. 37, Panel C). Concentrations of these metabolites were not significantly different from previous studies of perfused PC-3 cells, though acquired using nearly an order of magnitude less cells.

13

[00139] Hyperpolarized C MR Pyruvate Metabolism: Using this robust 5mm MR- compatible bioreactor, a small volume injection port was then interfaced for the rapid introduction of hyperpolarized (HP) substrates. HP Pyruvate was injected into varying densities of PC-3 cells and demonstrated high conversion to HP Lactate in real time (FIG. 37, Panel D). High SNR data (average max pyruvate SNR 433 ± 30) was easily obtained, using a 10° pulse, within seconds of the introduced HP Pyruvate and lasted nearly 2 min allowing for the capture of dynamics. The area under the curve of lactate, relative to the total carbon, was consistently less than 10% (0.068 ± 0.011) implying biochemical kinetics are in the first order regime. Normalizing to cell density, the generation of HP Lactate was proportional to the cell density and demonstrated a significant linear correlation (R =0.942). These numbers are significantly higher than that reported for living malignant prostate tissue slices in a much larger bioreactor (see, e.g., Keshari KR, et al. (2013) Metabolic

13

reprogramming and validation of hyperpolarized C lactate as a prostate cancer biomarker using a human prostate tissue slice culture bioreactor. The Prostate), but on the order of those in immortal cell cultures (see, e.g., Keshari KR, et al. (2010) Hyperpolarized (13)C spectroscopy and an NMR-compatible bioreactor system for the investigation of real-time cellular metabolism. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine 63(2):322-329; Harris T et al. (2009) Kinetics of hyperpolarized 13C1 -pyruvate transport and metabolism in living human breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America

106(43):18131-18136; and Day S et al. (2007) Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy. Nature medicine).

[00140] Extension to Glucose Uptake Using ¹⁸ F FDG PET: While the described

bioreactor system was designed for perfusion inside of a standard NMR

spectrometer, the extension of its use to other modalities is readily achievable. In order to compare metabolism of HP Pyruvate to uptake of glucose, the 5mm MR- compatible bioreactor was placed inside of the detector of a microPET and injected

18

with F-flurodeoxyglucose (FDG). In preliminary studies, FDG was injected into both an empty bioreactor and bioreactor filled with empty alginate encapsulates to assess delivery kinetics in the bioreactor (FIG 38). Empty alginate encapsulates demonstrated a similar release of FDG to free flowing medium, with both regions completely devoid of FDG signal 25 min after initiating the washout. This reaffirms that the encapsulates to not trap FDG, in preparation for cell studies.

[00141] In cell experiments, 5μ^ητΕ of FDG in medium was perfused through the bioreactor for 40 min and then washed out with FDG free medium. FIG. 39 demonstrates the simultaneous imaging of 4 bioreactors with increasing densities of

18

PC-3 encapsulated cells. At washout, the difference in trapping of F is readily observed and is linearly correlated to cell density (R =0.98, FIG. 39, Panel B).

[00142] Characterization of noninvasive biomarkers with response to NAMPT

targeting: One of the major strengths of this approach is the characterization of response to therapy using multi-modality noninvasive synergistic agents. While FDG provides the ability to assess changes in upstream metabolism by targeting glucose uptake through glucose like transporters (predominantly GLUT1) and

phosphorylation by hexokinase, HP Pyruvate can access downstream glycolysis at the level of conversion to lactate. Combining these probes can provide a better understanding of whether or not a drug hits its target and is effective. One new such target is the NAD⁺ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). NAMPT plays a role in a pathway utilized by cancer cells to reestablish steady state concentrations of NAD, which are depleted in functions such as poly(ADP-ribose)polymerase (PARP) activation. Since NAD is a critical cofactor in many biochemical processes, complete inhibition of synthesis would be catastrophic for the cell, in this instance the steady state pool is modulated via inhibition of its reestablishment, thus providing a novel means of targeting cancer cells.

[00143] With incubation of NAMPT inhibitor GEN617 (Genentech), a marked

decrease in conversion of HP pyruvate to lactate is observed (FIG. 40). This corresponded to a simultaneous decrease in 18F-FDG uptake. Decreased uptake of 18F-FDG is due to the inhibition of glycolysis since NAD is required for the downstream intermediate biochemical reactions. During this same period of time, 3 IP bioenergetics were not significantly different, implying preservation of cell health. ATP levels were confirmed ex vivo demonstrating no change within 50 hours of treatment. mRNA expression of relevant monocarboxylate transporters (MCT1 and MCT4) as well as lactate dehydrogenase (LDHA and LDHB) did not change significantly, as expected. Since the mechanism of action is the depletion of the cofactor NAD, levels of NAD(H) loss were directly correlated to a decrease in HP lactate. Additionally, with treatment an analogous increase in HP alanine was observed. This is a result of intracellular availability of HP pyruvate as a substrate, allowing alanine aminotransferase (ALT, EC) to dynamically compete with LDH. This provides both a negative and positive response to hitting the drug target and is quantitatively demonstrated using this approach.

[00144] Utilization for living Primary Human Prostate Tissue Slice cultures (TSC):

While immortal cell lines, such as PC-3 cells, are appropriate for the development of biomarker strategies they are not well suited for observing changes in metabolism and characterizing biomarker response, since their metabolism is radically different from that of the human prostate. Overall, the development and translation of novel therapeutics and biomarkers has been hindered by the lack of relevant models of disease. Recently, work in the field of tissue slice cultures has demonstrated that they may provide a relevant platform for investigation. For the purposes of imaging HP biomarkers, this requires the use small tissue cultures, which have been difficult to achieve previously. FIG. 41 demonstrates the characterization of human living 31 prostate tissue slice cultures (TSCs) in the 5mm bioreactor. In 4 slices (60μί), P bioenergetics analogous to significantly larger cultures (typically > lmL) were observed. These cultures remained viable for greater than 2 days, showing the robustness of the platform and its applicability to non-invasive monitoring (FIG. 41,

13

Panel A). This provided an appropriate platform for metabolic study and HP [1- C] pyruvate was injected. Conversion to HP Lactate was observed in real time as previously described, but here in a smaller culture (FIG. 41 , Panel B). The rate of formation and the magnitude was significantly lower than that of analogous PC-3 cells (10 fold lower, P<0.005), as normalized to β-ΝΤΡ. These levels are related to the lower levels of steady state lactate, previously observed when comparing prostate TSCs to immortal cell lines.

[00145] In addition to metabolism to lactate, HP C0₂ and bicarbonate formation in human tissue (FIG. 41 , Panel C) was observed for the first time. The rate of formation is on the order of lactate and demonstrates competing fluxes not observed in immortal prostate cancer cells. HP C0₂ is derived from metabolism of pyruvate through the pyruvate dehydrogenase complex (PDH, EC 1.2.4.1) and is then equilibrated with bicarbonate via carbonic anhydrase (CA, EC 4.2.1.1). This has been demonstrated in highly oxidative organs such as the murine heart and liver, but never in living human tissues. The ratio of bicarbonate to C0₂ is indicative of the intracellular pH and is governed by the Henderson-Hasselbach equation.

Interestingly the ratio of bicarbonate to C0₂ does not instantly equilibrate on the timescale of the HP experiment in prostate tissue, implying the activity of carbonic anhydrase is not sufficient, but easily assessed during the experiment.

[00146] Given the rate of incorporation of pyruvate into the TCA cycle, indicated by

13

the observed bicarbonate, HP [1,2- C₂] pyruvate was then injected into living TSCs. When the 3 carbon molecule of pyruvate is metabolized, the C 1 is predominantly cleaved by PDH in the prostate and released as C0₂. The C2 then enters through acetyl-coA and through TCA cycle flux can label the C5 of glutamate. This is demonstrated in (FIG. 41, Panel C), where simultaneous HP labeling of glutamate, lactate and bicarbonate are easily identified, indicative of multiple fluxes in time. The prostate is known to be highly glycolytic and to conduct high rates of oxidative metabolism and this is recapitulated in human living prostate TSCs. Formation of C2 glutamate was observed to be more rapid and to higher magnitude than bicarbonate (FIG. 41). This implies that post enzymatic conversion by PDH, TCA flux and equilibration to glutamate by glutamate dehydrogenase (GLDH, EC 1.4.1.2) is potentially faster than carbonic anhydrase activity in the prostate. It is important to note that these real time metabolic investigations are extremely difficult using previously existing means, but are readily observed using the in living human tissues using the devices and systems described herein.

Claims

CLAIMS What is claimed is:

1. A bioreactor system comprising:

a concentric exchanger comprising

2. The bioreactor system of claim 1 , wherein the third elongate structure comprises an external insulation layer.

3. The bioreactor system of claim 1 or 2, wherein the third elongate structure is

fluidically coupled to a temperature-controlled fluid flow.

4. The bioreactor system of claim 3, wherein the temperature-controlled fluid flow is a temperature-controlled water flow.

5. The bioreactor system of any one of claims 1 to 4, wherein the second elongate

structure is fluidically coupled to a pressurized gas input.

6. The bioreactor system of any one of claims 1 to 5, wherein the first elongate structure is fluidically coupled to a perfusion media input.

7. The bioreactor system of any one of claims 1 to 6, wherein one or more of the first, second and third elongate structures are tubular structures.

8. The bioreactor system of any one of claims 1 to 7, wherein the third elongate structure comprises a flexible polymer material.

9. The bioreactor system of claim 8, wherein the third elongate structure comprises an external insulation layer.

10. The bioreactor system of any one of claims 1 to 9, wherein the second elongate

structure comprises a flexible polymer material.

11. The bioreactor system of claim 10, wherein the flexible polymer material comprises one or more of a fluorinated ethylene propylene (FEP), a polytetrafluoroethylene (PTFE), and a perf uoroalkoxy (PFA).

12. The bioreactor system of claim 10, wherein the flexible polymer material is relatively gas impermeable.

13. The bioreactor system of any one of claims 1 to 12, wherein the first elongate

structure comprises a flexible polymer material.

14. The bioreactor system of claim 13, wherein the flexible polymer materials is

relatively gas permeable.

15. The bioreactor system of claim 13, wherein the flexible polymer material comprises a silicone.

16. The bioreactor system of any one of claims 1 to 15, wherein the bioreactor comprises a bioreactor construct comprising a UV-cured polymer material.

17. The bioreactor system of any one of claims 1 to 16, wherein the bioreactor comprises a bioreactor construct comprising a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

18. The bioreactor system of claim 17, wherein the bioreactor construct comprises a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water.

19. The bioreactor system of any one of claims 1 to 16, wherein the bioreactor comprises a bioreactor construct comprising a magnetic resonance (MR) compatible material.

20. The bioreactor system of any one of claims 1 to 19, wherein the bioreactor comprises a bioreactor construct sized to fit within an approximately 5 mm outer diameter tube.

21. The bioreactor system of any one of claims 1 to 20, wherein the bioreactor comprises a bioreactor construct sized to fit within an approximately 4 mm inner diameter tube.

22. The bioreactor system of any one of claims 1 to 20, wherein the bioreactor comprises a bioreactor construct having a diameter of about 4 mm.

23. The bioreactor system of any one of claims 1 to 20, wherein the bioreactor comprises a bioreactor construct having a diameter of less than 4 mm.

24. The bioreactor system of any one of claims 1 to 23, wherein the bioreactor comprises a bioreactor construct comprising a first end and a second end and defines a central bore extending from the first end to the second end, wherein the central bore is configured to fluidically receive the input from the first bore of the first elongate structure.

25. The bioreactor system of claim 24, wherein the central bore has a diameter of from about 1 mm to about 2 mm.

26. The bioreactor system of claim 24, wherein the central bore has a diameter of from about 0.5 mm to about 1 mm.

27. The bioreactor system of claim 24 or 25, wherein the bioreactor construct comprises a central shaft defining the central bore.

28. The bioreactor system of claim 27, wherein the bioreactor construct comprises a plurality of sidewalls extending the length of the bioreactor construct and defining sidewall openings in the bioreactor construct.

29. The bioreactor system of claim 28, wherein the bioreactor construct comprises a plurality of struts extending perpendicularly from the central shaft and connecting the central shaft with the plurality of sidewalls.

30. The bioreactor system of any one of claims 27 to 29, wherein the first end and the second end of the bioreactor construct each define a plurality of openings which, when the bioreactor construct is positioned in a tube, provide a flow path between the first end, the sidewall openings and the second end.

31. The bioreactor system of any one of claims 27 to 30, wherein the diameter of the central bore is greater at the first end of the bioreactor construct and the second end of the bioreactor construct than at a midpoint along the central bore.

32. The bioreactor system of any one of claims 27 to 31, wherein the first end and the second end of the bioreactor construct each comprise a plurality of ridges extending at least partially along the length of the bioreactor construct.

33. The bioreactor system of claim 24 or 25, wherein the bioreactor construct defines a plurality of through holes positioned externally to the central bore and extending from the first end to the second end.

34. The bioreactor system of claim 33, wherein the through holes are positioned radially around the central bore.

35. The bioreactor system of claim 33 or claim 34, wherein the through holes extend parallel to the central bore.

36. The bioreactor system of any one of claims 33 to 35, wherein the through holes have a diameter of from about 0.2 mm to about 0.6 mm.

37. The bioreactor system of claim 36, wherein the through holes have a diameter of about 0.4 mm.

38. The bioreactor system of claim 36, wherein the through holes have a diameter of about 300 μιη.

39. The bioreactor system of any one of claims 33 to 37, wherein the length of the

bioreactor construct from the first end to the second end is from about 2 mm to about 6 mm.

40. The bioreactor system of claim 39, wherein the length of the bioreactor construct from the first end to the second end is about 3 mm.

41. The bioreactor system of any one of claims 1 to 39, comprising a first manifold coupled to the concentric exchanger at a first end of the concentric exchanger, the first manifold comprising a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports, and wherein the first manifold provides a flow path between the third bore of the third elongate structure and the third port.

42. The bioreactor system of claim 41 , wherein an external wall of the first port engages the third bore of the third elongate structure via press-fit engagement.

43. The bioreactor system of claim 42, wherein the external wall of the first port is sealed against the third bore of the third elongate structure with an adhesive.

44. The bioreactor system of claim 42, wherein the first port comprises external barbs or ridges to effect the press-fit engagement.

45. The bioreactor system of any one of claims 41 to 44, comprising a seal positioned in the second port between the body of the manifold and the second elongate structure.

46. The bioreactor system of any one of claims 41 to 45, comprising a second manifold coupled to the concentric exchanger at a second end of the concentric exchanger, the second manifold comprising a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port of the second manifold interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports of the second manifold, and wherein the second manifold provides a flow path between the third bore of the third elongate structure and the third port of the second manifold.

47. The bioreactor system of claim 46, wherein an external wall of the first port of the second manifold engages the third bore of the third elongate structure via press-fit engagement.

48. The bioreactor system of claim 47, wherein the external wall of the first port is sealed against the third bore of the third elongate structure with an adhesive.

49. The bioreactor system of claim 47, wherein the first port of the second manifold comprises external barbs or ridges to effect the press-fit engagement.

50. The bioreactor system of any one of claims 46 to 49, comprising a seal positioned in the second port of the second manifold between the body of the second manifold and the second elongate structure.

51. The bioreactor system of any one of claims 41 to 50, wherein the first and/or second manifold comprises a UV-cured polymer material.

52. The bioreactor system of any one of claims 41 to 51, wherein the first and/or second manifold comprises a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

53. The bioreactor system of claim 52, wherein the first and/or second manifold

comprises a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water.

54. The bioreactor system of any one of claims 41 to 51, wherein the first and/or second manifold comprises a magnetic resonance (MR) compatible material.

55. The bioreactor system of any one of claims 46 to 54, wherein the third port of the first manifold functions as an inlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an outlet port for the temperature- controlled fluid flow.

56. The bioreactor system of any one of claims 46 to 54, wherein the third port of the first manifold functions as an outlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an inlet port for the temperature- controlled fluid flow.

57. The bioreactor system of any one of claims 1 to 56, comprising a perfusion media reservoir coupled to the first elongate structure.

58. The bioreactor system of claim 57, wherein the perfusion media reservoir receives perfusion media after it has passed through the bioreactor.

59. The bioreactor system of claim 57 or 58, comprising a pump configured to pump perfusion media from the perfusion media reservoir through the first bore of the first elongate structure.

60. The bioreactor system of claim 59, wherein the pump is a peristaltic pump.

61. The bioreactor system of any one of claims 1 to 60, comprising an injection port coupled to the first elongate structure.

62. The bioreactor system of any one of claims 1 to 61, comprising a pressurized gas reservoir coupled to the second elongate structure.

63. The bioreactor system of any one of claims 1 to 62, wherein the bioreactor is

positioned in a tube.

64. The bioreactor system of claim 63, wherein the tube is an NMR tube.

65. The bioreactor system of any one of claims 1 to 64, comprising a tube cap, the tube cap comprising a body defining an inlet port, an outlet port, a tube-engagement opening, and an internal chamber connecting the inlet port, the outlet port and the tube engagement opening.

66. The bioreactor system of claim 65, wherein the body of the tube cap comprises an internal wall at least partially defining the internal chamber, and wherein the internal wall defines a channel configured to facilitate liquid flow through the internal chamber and out the outlet port.

67. The bioreactor system of claim 66, wherein the channel has a spiral structure.

68. The bioreactor system of claims 65 to 67, wherein the tube cap comprises a UV- cured polymer material.

69. The bioreactor system of any one of claims 65 to 68, wherein the tube cap comprises a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

70. The bioreactor system of claim 69, wherein the tube cap comprises a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water.

71. The bioreactor system of any one of claims 65 to 68, wherein the tube cap comprises a magnetic resonance (MR) compatible material.

72. The bioreactor system of any one of claims 65 to 71 , wherein the tube cap comprises one or more O-rings positioned in the internal chamber so as to engage an external wall of a tube to which the tube cap is engaged.

73. The bioreactor system of any one of claims 65 to 72, comprising one or more

connectors which, individually or together, provide a fluid flow path from the first bore of the first elongate structure of the concentric exchanger through the inlet port of the tube cap to the bioreactor.

74. The bioreactor system of any one of claims 65 to 73, wherein the outlet port of the tube cap receives perfusion media after it has passed through the bioreactor.

75. The bioreactor system of claim 74, wherein the perfusion media reservoir is

f uidically connected to the outlet port of the tube cap.

76. A bioreactor comprising:

a first end;

a second end;

77. The bioreactor of claim 76, wherein the bioreactor comprises a plurality of struts extending perpendicularly from the central shaft and connecting the central shaft with the plurality of sidewalls.

78. The bioreactor of claim 76 or 77, wherein the first end and the second end of the bioreactor each define a plurality of openings which, when the bioreactor is positioned in a tube, provide a flow path between the first end, the sidewall openings and the second end.

79. The bioreactor of any one of claims 76 to 78, wherein the diameter of the central bore is greater at the first end of the bioreactor and the second end of the bioreactor than at a midpoint along the central bore.

80. The bioreactor of any one of claims 76 to 79, wherein the first end and the second end of the bioreactor each comprise a plurality of ridges extending at least partially along the length of the bioreactor.

81. The bioreactor of any one of claims 76 to 80, wherein the bioreactor comprises a UV-cured polymer material.

82. The bioreactor of any one of claims 76 to 81, wherein the bioreactor comprises a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

83. The bioreactor of claim 82, wherein the bioreactor comprises a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water.

84. The bioreactor of any one of claims 76 to 81, wherein the bioreactor comprises a magnetic resonance (MR) compatible material.

85. The bioreactor of any one of claims 76 to 84, wherein the bioreactor is sized to fit within an approximately 5 mm outer diameter tube.

86. The bioreactor of any one of claims 76 to 85, wherein the bioreactor is sized to fit within an approximately 4 mm inner diameter tube.

87. The bioreactor of any one of claims 76 to 85, wherein the bioreactor has a diameter of about 4 mm.

88. The bioreactor of any one of claims 76 to 85, wherein the bioreactor has a diameter of less than 4 mm.

89. A concentric exchanger comprising:

a second elongate structure defining a second bore extending lengthwise therethrough; a third elongate structure defining a third bore extending lengthwise therethrough, wherein the first elongate structure is positioned concentrically within the second bore of the second elongate structure and the second elongate structure is positioned concentrically within the third bore of the third elongate structure; and a first manifold coupled to the concentric exchanger at a first end of the concentric exchanger, the first manifold comprising a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports, and wherein the first manifold provides a flow path between the third bore of the third elongate structure and the third port.

90. The concentric exchanger of claim 89, wherein the third elongate structure comprises an external insulation layer.

91. The concentric exchanger of claim 89 or 90, wherein the third elongate structure is f uidically coupled to a temperature-controlled fluid flow.

92. The concentric exchanger of claim 91 , wherein the temperature-controlled fluid flow is a temperature-controlled water flow.

93. The concentric exchanger of any one of claims 89to 92, wherein the second elongate structure is fluidically coupled to a pressurized gas input.

94. The concentric exchanger of any one of claims 89to 93, wherein the first elongate structure is fluidically coupled to a perfusion media input.

95. The concentric exchanger of any one of claims 89 to 94, wherein one or more of the first, second and third elongate structures are tubular structures.

96. The concentric exchanger of any one of claims 89 to 95, wherein the third elongate structure comprises a flexible polymer material.

97. The concentric exchanger of any one of claims 89 to 96, wherein the second elongate structure comprises a flexible polymer material.

98. The concentric exchanger of claim 97, wherein the flexible polymer material comprises one or more of a fluorinated ethylene propylene (FEP), a

polytetrafluoroethylene (PTFE), and a perfluoroalkoxy (PFA).

99. The concentric exchanger of any one of claims 89 to 98, wherein the first elongate structure comprises a flexible polymer material.

100. The concentric exchanger of claim 99, wherein the flexible polymer material comprises a silicone.

101. The concentric exchanger of claim 100, wherein an external wall of the first port engages the third bore of the third elongate structure via press-fit engagement.

102. The concentric exchanger of claim 101, wherein the first port comprises external barbs or ridges to effect the press-fit engagement.

103. The concentric exchanger of any one of claims 89 to 102, comprising a seal positioned in the second port between the body of the manifold and the second elongate structure.

104. The concentric exchanger of any one of claims 89 to 103, comprising a

second manifold coupled to the concentric exchanger at a second end of the concentric exchanger, the second manifold comprising a body defining an internal chamber and first, second and third ports connected via the internal chamber, wherein the first port of the second manifold interfaces with the third bore of the third elongate structure, the first and second elongate structures extend through the first and second ports of the second manifold, and wherein the second manifold provides a flow path between the third bore of the third elongate structure and the third port of the second manifold.

105. The concentric exchanger of claim 104, wherein an external wall of the first port of the second manifold engages the third bore of the third elongate structure via press-fit engagement.

106. The concentric exchanger of claim 105, wherein the external wall of the first port of the second manifold is sealed against the third bore of the third elongate structure with an adhesive.

107. The concentric exchanger of claim 105, wherein the first port of the second manifold comprises external barbs or ridges to effect the press-fit engagement.

108. The concentric exchanger of any one of claims 104 to 107, comprising a seal positioned in the second port of the second manifold between the body of the second manifold and the second elongate structure.

109. The concentric exchanger of any one of claims 89 to 108, wherein the first and/or second manifold comprises a UV-cured polymer material.

110. The concentric exchanger of any one of claims 89 to 109, wherein the first and/or second manifold comprises a material having a magnetic susceptibility within 10% of the magnetic susceptibility of water.

111. The concentric exchanger of claim 1 10, wherein the first and/or second

manifold comprises a material having a magnetic susceptibility within 5% of the magnetic susceptibility of water.

112. The concentric exchanger of any one of claims 89 to 109, wherein the first and/or second manifold comprises a magnetic resonance (MR) compatible material.

113. The concentric exchanger of any one of claims 104 to 112, wherein the third port of the first manifold functions as an inlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an outlet port for the temperature-controlled fluid flow.

114. The concentric exchanger of any one of claims 104 to 112, wherein the third port of the first manifold functions as an outlet port for a temperature-controlled fluid flow and the third port of the second manifold functions as an inlet port for the temperature-controlled fluid flow.

115. The concentric exchanger of any one of claims 89 to 114, comprising a

perfusion media reservoir coupled to the first elongate structure.

116. The concentric exchanger of claim 115, comprising a pump configured to pump perfusion media from the perfusion media reservoir through the first bore of the first elongate structure.

117. The concentric exchanger of claim 1 16, wherein the pump is a peristaltic pump.

118. The concentric exchanger of any one of claims 89 to 117, comprising an injection port coupled to the first elongate structure.

119. The concentric exchanger of any one of claims 89 to 118, comprising a pressurized gas reservoir coupled to the second elongate structure.

120. A method of perfusing cells and/or tissues, the method comprising

positioning cells and/or tissues in a bioreactor of the bioreactor system of any one of claims 1-75; and

121. The method of claim 120, wherein the method further comprises perfusing the cells and/or tissues while the bioreactor is positioned in an NMR tube in the bore of an NMR spectrometer.

122. The method of claim 121, wherein the method further comprises analyzing the cells and/or tissues using NMR spectroscopy.