This research was funded by grants from the National Institutes of Health (R01 GM148741 I.R.S.), U01 EY034591, R01 EY034364, BrightFocus Foundation, Research to Prevent Blindness (J.R.C.) and R01 EY006641, R01 EY017863 and R21 EY032597 (J.B.H.).

Pumpless Multi-Channel Flow Culture to Maintain and Assess Tissues and Cells

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I.R.S., M.G., and K.B. have financial ties to EnTox Sciences, Inc. (Mercer Island, WA), the manufacturer/distributor of the BaroFuse perifusion system described in this study. All the other authors declare no conflicts of interest.

Due to the importance of bioenergetics in all aspects of cell function and maintenance of various components of the eye, there is a critical need for methods to study its regulation. In particular, neural retina and RPE depend on metabolism for both generation of energy as well as intra- and inter-cellular signaling14,15,16,17. Because of their high oxidative capacity, isolated tissues of the eye are not well-maintained under static conditions18,19 and therefore study of isolated components of the eye require flow systems that can both maintain and assess metabolic processes. The fluidics system was developed to generate OCR and LPR data from a wide range of tissue types and in this paper we presented detailed protocols that were found to produce optimal results.
The major determinant for generating robust data using the flow system includes pre-equilibration of CO2-based media/buffer at 39 &#176;C (to ensure perifusate is not supersaturated with dissolved gas that would degas during the experiment). In particular, media or KRB buffer stored at 4&#160;&#176;C will be supersaturated relative to 37 &#176;C and will degas during the experiment if pre-equilibration times are insufficient. In addition, tissue loaded into the tissue chambers must not be traumatized by improper isolation of tissue due to tearing or incomplete separation of tissue, or by exposing tissue in low amount of bicarbonate-based buffer to atmospheric air for too long. The temperature control, flow stability and reliability of O2 detection have little variability and these factors do not contribute significantly to failure rate.
The instrument has eight flow channels/tissue chambers that run simultaneously which are supplied with perifusate from two reservoirs, four tissue chambers for each reservoir. To get the most accurate time-courses of OCR, kinetic curves are baseline corrected by chambers that are not loaded with tissue. Thus, a typical experimental protocol would involve two groups of three tissue chambers. Protocols in general fall into two categories: one is the different test compound protocols on each side (for instance drug/vehicle on one side of the MRM, and just vehicle on the other); the second is same test compound injection protocol on both sides of the MRM, but different tissue or tissue model on each side of the MRM. In this paper, the effects of oligomycin and FCCP on retina were compared to OCR by tissue that were not exposed to any test compounds, and two tissues were concomitantly assessed under the same protocol and conditions to identify tissue-specific behavior. The latter was illustrated in this study by showing increased dynamic range of metabolic rate by RPE-choroid-sclera relative to retina in parallel in the same experiment. Other reports have described a wider range of study designs including measuring the effects varying O2 levels on OCR and LPR, and concentration dependencies of fuels, drugs, and toxins20,21. In addition, although we have limited the analysis of outflow fractions to the measurement of lactate and calculation of LPR, the information content of an experiment increases greatly if multiple compounds and classes of compounds in the outflow fractions are assayed such as hormones, neurotransmitters, cell signals, and metabolites that can exit the cells20,22,23.
The loading of isolated retina or RPE-choroid-sclera is straightforward, and once isolated these tissues are simply placed into the top of the tissue chambers with forceps and allowed to sink down to the frit. RPE cells cultured on filter inserts develop appropriate polarization and markers of RPE maturity after 4-8 weeks in culture. It is not feasible to remove the RPE for live cell analysis once attached to the transwell membrane, if RPE maturity and polarization are to be maintained24. The perifusion chamber can accommodate strips of the transwell membrane that are cut with a scalpel while submerged in buffer and rapidly inserted into the tissue chambers. Although cutting filter strips has been placed into a static system24, no other fluidics method to assess these important cell types are available. The responses of RPE cells were rapid and more dynamic than either the retina or the RPE-choroid-sclera, likely in part due to immediate access of both the apical and basal aspects of the RPE cells configured as a monolayer on the membrane insert.
Another factor in assuring data has the highest signal to noise is selecting the optimal ratio of tissue loaded into the perifusion chambers relative to the flow rate. Too little tissue relative to the flow rate results in a difference of dissolved O2 concentration between inflow and outflow that is very small and difficult to measure reliably. In contrast, if the flow is too slow, then the concentration of O2 becomes so low that the tissue is affected by hypoxia. Nonetheless, the gas pressure-driven liquid flow can be maintained at flow rates down to 5 mL/min requiring only small amounts of tissue for accurate OCR and LPR measurements. In the experiments shown here, about 20 mL/min/channel was used which was suitable for either one retina, two RPE-choroid-scleras, or 360,000 RPE cells. To minimize the system effects that delay and disperse the exposure of the tissue to the injected test compound, multiple sizes of the tissue chambers are supplied, so that the amount of tissue (and flow rate) is matched with the appropriate size of the chamber.
Data from the analyses shown in this paper were represented in two ways: absolute magnitude with respect to rate, or fractional changes relative to a steady-state or baseline. The focus was on illustration of measurement of responses to test compounds. However, the fluidics system is well-suited to assess and compare effects of tissue treatment prior to the perifusion analysis such as genetic modifications. Testing whether a treatment is different from control is most robust if the effects of the treatment on normalized responses of test compounds is analyzed. If the analysis requires absolute magnitudes, the statistical power of the analyses of specimens that are pretreated is maximized if their assessment and controls are carried out in the same perifusion experiment.
Except for the stirrer, all parts that come in contact with liquid are supplied by the manufacturer as consumables and have been sterilized. These parts should not be reused, as experiments will occasionally be lost due to incomplete cleaning and contaminated surfaces. The system at the outset of setup is sterile. However, media is added to the MRM, and tissue is loaded in the chambers under non-sterile conditions. We have measured OCR in the system that is assembled with parts that are sterile, but where the experiment itself is carried out under non-sterile conditions. It takes about 14 h for bacteria to accumulate to the point of having measurable OCR (unpublished results). If protocols are used that are less than 10 h or so, then accumulation of bacteria and any effects due to these will be negligible.
Many investigators use instruments that are designed to measure OCR under static incubation of a monolayer of cells with a relatively high throughput25,26. In contrast, the fluidics instrument we have tested and described in this paper maintains tissue by ensuring adequate O2 delivery which is critical for the greater diffusion distances that are present in tissue specimens. In addition, it is able to collect fractions allowing assessment of multiple parameters in parallel with OCR which greatly enhances the ability to study relationships between them. Finally, dissolved gas concentrations (such as O2 and CO2) can be controlled, increasing the duration of experiments with bicarbonate-based media and buffer, enabling the user to study the effects of O2. It should be pointed out, a limitation for both methodologies is the inability to study the washout of test compounds, a functionality that other perifusion systems have4,27,28. Another consideration when determining the optimal analysis modality is the fact that fluidics systems use more media and test compounds than static systems. The extra expense is minimized with the current fluidics systems though due to the low flow rates that the system can be used.
Overall, a detailed description of the protocols to perform experiments with a new flow/assessment instrument is described. Data generated with retina and RPE-choroid-sclera recapitulated previous results obtained with systems that are much more difficult to use (and not readily available). It was also demonstrated that the system can maintain and assess RPE cells attached to transwell membranes, a very important cellular model that has not previously been analyzed with flow systems due to the fragility of the cells. The main parts of the protocol consist of a 75 min setup time, followed by a 90 min equilibration period and the experimental protocol making it suitable for routine use by laboratories that do not specialize in the operation of fluidics systems. Although we focused on measuring the acute response of tissue to test compounds, the system is very suitable to comparing tissue from various sources such as animal models or cells models that have been genetically altered or undergone test treatments/conditions. In addition, the scope of assays that can be conducted on the outflow fractions are wide-ranging and include metabolites, cell signaling molecules and secreted hormones/neurotransmitters as well as multi-component analysis generated by mass spectrometry on the fractions as well as the tissue.

Perifusion systems have a long history in the life sciences. In particular, for the study of secretory function by islets, they have been used to characterize the kinetics of insulin secretion in response to secretagogues1. In addition to collecting outflow fractions for subsequent assay of hormones and metabolites, real-time sensors have been incorporated, predominantly for the detection of oxygen consumption2,3,4. Widespread efforts to better understand mechanisms mediating diseases of the eye have been limited by a lack of physiologically relevant methods to assess metabolic regulation and dysregulation of the various isolated components of the eye, including the retina, retinal pigment epithelial (RPE)-choroid-sclera and cultured RPE cells. Static systems designed for cultured cells have been adapted for tissue5, but tissue requires flow for adequate oxygenation. Flow systems have been successful at accurately and reproducibly measuring real-time responses in oxygen consumption rate (OCR) by the retina and RPE-choroid-sclera, and the tissues stay metabolically stable for more than 8 h allowing highly informative protocols involving multiple test compounds4,6,7,8,9. Nonetheless, the operation of fluidics systems has historically required a custom-made apparatus and trained technical staff in non-standardized methodologies. Such systems have not been adopted as the standard methodology in most laboratories. The BaroFuse is a newly developed fluidics system that does not rely on pumps, but rather on gas pressure to drive flow through multiple channels and tissue chambers (Figure 1). Each channel is continuously monitored for OCR, and the outflow is collected with a plate-based fraction collector for subsequent assay of contents. Importantly, the tissue perifusion chambers for the instrument are designed to accommodate tissues of various geometries and sizes.
The heart of the instrument is the fluidics system, where flow is driven from a sealed, pressurized reservoir through small inner diameter (ID) tubing (contributing the most significant flow resistance in the fluid circuit) up into the glass tissue chambers that house the tissue. Pressure to the media reservoir module (MRM) is supplied by low-pressure and high-pressure regulators connected to a gas cylinder containing a mixture of gases (typically 21% O2, 5% CO2, balance N2), and the reservoir is sealed from the top by the perifusion chamber module (PCM) that holds the tissue chamber assemblies (TCAs). Flow rate is controlled by the length and ID of the resistance tubes and the pressure setting of a low-pressure regulator. Outflow tubes connected to the top of tissue chambers deliver fluid to either a waste receptacle (that is continuously weighed for automatic determination of flow rate) or into wells of a 96-well plate controlled by the fraction collector. The O2 detection system measures the lifetime of an O2-sensitive dye painted on the inside of each of the glass tissue chambers downstream of the tissue. This information is then used to continuously calculate OCR. The entire fluidics system resides in a temperature-controlled enclosure and the gas tank, fraction collector and computer are the major components of the instrument (Figure 2A). Finally, software that runs the instrument serves to control its operation (including the preparation and timing of injected test&#160;compounds, flow measurement system, and fraction collector timing), as well as processing and graphing the OCR data and other supplemental measurements.
In this paper we describe the protocols for using the fluidics system to perifuse and assess OCR and lactate production rate (LPR) for various isolated components of the eye. LPR is a parameter reflecting glycolytic rate that is highly complementary to OCR, where the pair accounts for the two major branches of energy generation from carbohydrates in the cell10. As preparation of the tissue and loading it into the tissue chambers is best learned by watching the procedure, the video will help illustrate several of the critical steps that are performed during set up and operation that are not easily conveyed by text alone.
The description of the protocol is divided up into 8 sections that correspond to different phases of the experiment (Figure 2B): 1. pre-experimental preparation; 2. preparation/equilibration of the perifusate; 3. instrument set up; 4. tissue equilibration; 5. experimental protocol; 6. instrument break down; 7. data processing; and 8. assays of outflow fractions.

<table><tbody><tr><td>&lt;strong&gt;BIOLOGICAL SAMPLES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C57BL/6J mice</td><td>Envigo Harlan (Indianapolis, IN)</td><td>N/A</td><td></td></tr><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FCCP</td><td>Sigma-Aldrich</td><td>C2920L9795</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270G</td><td></td></tr><tr><td>KCN</td><td>Sigma-Aldrich</td><td>60178</td><td></td></tr><tr><td>Lactate&amp;nbsp;</td><td>MilliporeSigma</td><td>L6661</td><td></td></tr><tr><td>Oliigomycin A</td><td>Sigma-Aldrich</td><td>75351L9795</td><td></td></tr><tr><td>&lt;strong&gt;CELL CULTURE AND TISSUE HARVESTING&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beuthanasia-D</td><td>Schering-Plough Animal Health Corp., Union, NJ</td><td>N/A</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A3059</td><td></td></tr><tr><td>Euthasol, 390 mg/ml sodium pentobarbital</td><td>Virbac</td><td>RXEUTHASOL</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich&amp;nbsp;</td><td>12303C</td><td></td></tr><tr><td>Hank&amp;rsquo;s Buffered Salt Solution</td><td>GIBCO</td><td>14065056</td><td></td></tr><tr><td>Krebs Ringer Bicarbonate (KRB)</td><td>Thermo Fisher Scientific</td><td>J67795L9795</td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>#CB-40230</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>ROCKi&amp;nbsp;</td><td>Selleck Chemicals</td><td>Y-27632</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>ThermoFisher</td><td>#25-200-072</td><td></td></tr><tr><td>&lt;strong&gt;SUPPLIES&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gas Cylinders: 21% O2/5% CO2/balance N2</td><td>Praxair Distribution, Inc</td><td>N/A</td><td></td></tr><tr><td>Transwell filters&amp;nbsp;</td><td>MilliporeSigma</td><td>3470</td><td></td></tr><tr><td>&lt;strong&gt;COMMERCIAL ASSAYS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amplex Red Glucose/Glucose Oxidase Assay Kit</td><td>ThermoFisher</td><td>A22189</td><td></td></tr><tr><td>Glucose Oxidase from &lt;em&gt;Aerococcus viridans&lt;/em&gt;</td><td>Invitrogen (Carlsbad, CA)</td><td>A22189L9795</td><td></td></tr><tr><td>Lactate Oxidase</td><td>Sigma-Aldrich</td><td>L9795</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BaroFuse&amp;nbsp; Multi-Channel Perifusion system</td><td>EnTox Sciences, Inc (Mercer Island, WA</td><td>Model 001-08</td><td></td></tr><tr><td>Synergy 4 Fluorometer</td><td>BioTek (Winooski, VT)</td><td>S4MLFPTA</td><td></td></tr></tbody></table>

maintaining and assessing various tissue and cell types of the eye using a novel pumpless fluidics system

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

To illustrate the resolution of the data generated from isolated components of the eye, OCR and LPR was measured with three types of tissue (retina, RPE-choroid-sclera, and RPE cells) following a commonly used protocol (the mitochondrial stress test10; Figure 10, Figure 11, and Figure 12). The amount of tissue used for each tissue is shown in Table 1. Data was processed and graphed using the software package that was developed for the fluidics system. The preparation of retina and RPE-choroid-sclera is relatively straight-forward and takes less than 20 min for each tissue type. OCR was constant during the time that test compounds were injected, indicating stable health and function of the tissue and supporting the validity of the method (Figure 10). Once validated for each tissue type, we have not found it necessary to run controls where no test compounds are injected for each experiment. Consistent with data obtained using more conventional perifusion methods6,8,13, OCR decrease in response to oligomycin and increased OCR in response to FCCP. Changes in LPR were in the opposite direction of those observed for OCR: oligomycin increased LPR, which then decreased (but only slightly) in response to FCCP (Figure 11). To compare the statistical significance of the effect of each sequential test compound, t-tests were performed (which are calculated automatically by the software that comes with the instrument). Since the goal of the paper was to describe how to perform the method, the number of replicates carried was not always high enough to produce statistical significance. In general, though, when the number of replicates were 3 or more, effects of FCCP and oligomycin on both OCR and LPR were significant.
RPE cells have not been previously analyzed with flow systems but responded similarly to RPE-choroid-sclera (consistent with the view that a large fraction of OCR is due to RPE cells; Figure 11). These illustrative examples highlight ability of the system to maintain tissue viability as reflected by the stability of OCR in the control channels, and the high signal to noise ratio for changes in OCR of the magnitude induced by oligomycin and FCCP, which was more than 100 to 1. In addition, assays of outflow fractions can be used to correlate the rate of uptake or production of a wide array of compounds exchanging with the extracellular fluid are complementary to OCR (in this case, LPR). These features of the instrument allowed accurate quantification of characteristic differences in tissue responses between tissue types performed in parallel. OCR by RPE-choroid-sclera and RPE cells are consistently more sensitive to oligomycin than retina (Figure 11 and Figure 12), although for the RPE-choroid-sclera the duration of exposure to FCCP was not long enough to reach steady state. A point to consider arose when using DMSO as a solvent. At higher concentrations, (0.2%) DMSO had a transient effect on OCR by retina (presumably reflecting an effect of a change in osmotic pressure brought about by DMSO&#39;s effect on membrane permeability).
Based on the assumption that KCN completely inhibits respiration by its direct action on cytochrome c oxidase, OCR at the end of the KCN exposure is set to 0 and all OCR values are calculated based on the change relative to the KCN value. OCR can occur independent of the respiratory chain and cytochrome c oxidase. However, the magnitude of this contribution to overall OCR is generally not more than a few percent (data not shown) and the extended length of time that tissue is exposed to KCN ensures that substrates of oxidases that are not part of the electron transport chain have been depleted.
Statistical analysis 
Single experiments were shown as indicated in the figures, but with multiple channels that were averaged. Data was then graphed as the average &#177; the standard error (SE; calculated as SD/&#8730;n).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65399/65399fig1v2.jpg" /> 
Figure 1.&#160;Schematic of the fluidics/assessment system. Major components include the enclosure, temperature control elements, fluidics and tissue chamber systems, regulation of gas pressure in the head space above perifusate, fraction collector/flow rate monitoring, and O2 detectors. Abbreviations: MRM = Media Reservoir Module, PCM = Perifusion Chamber Module, TCA= Tissue Chamber Assemblies. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65399/65399fig02.jpg" /> 
Figure 2. (A)&#160;Picture of the major components of the instrument. The major components consist of gas tank (pressure regulators), enclosure, fraction collector and computer. (B)&#160;Experimental flow chart showing the major categories of steps and the time it takes to complete them. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65399/65399fig03.jpg" /> 
Figure 3. View of the MRM. The MRM is shown with an MRM insert (left) and stir bars (right) placed into the bottom of the MRM inserts (placed in each side of the MRM Divider). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65399/65399fig04.jpg" /> 
Figure 4. Tubing assembly and purge tubing assembly in the MRM. (A)&#160;Test compound injection tubing assembly and purge tubing assembly attached to ports on the MRM. (B-C) The test compound injection assembly and purge tubing assembly (B) are placed in the groove in&#160;the front of Enclosure (C). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65399/65399fig05.jpg" /> 
Figure 5. Powering up the MRM temperature controller. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65399/65399fig06.jpg" /> 
Figure 6. Tissue chambers and gas tank.&#160;Positioning the O2 detector on the detector stand (which also supports the MRM and PCM), and placement of the band around the fins of the PCM that help secure the tissue chambers in place. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65399/65399fig07.jpg" /> 
Figure 7. (A) High- and low-pressure regulators on the gas tank. (B-C) Purge tube. Purge tube allows the headspace in the MRM to clear of air and fill with gas from the supply tank. Pictures showing open purge tube (B) and close purge tube (C). Test compound injection assembly stays closed through the purge process. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65399/65399fig08v3.jpg" /> 
Figure 8.&#160;Tissue chamber and the outflow setup. (A) Dimensions of the Transwell membrane after it is cut into three strips of equal width. (B) Outflow multi-tube support. (C) Outflow multi-tube support positioned on the lip of the enclosure with the tubing adapters near the tissue chambers. (D) Picture of outflow tubing assemblies attached to the tissue chambers. (E)&#160;Aerial view of the enclosure without the Lid. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig08largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65399/65399fig09.jpg" /> 
Figure 9. Injection of compound in MRM. Injecting a test compound through the injection port into the MRM using a 5 mL syringe. <a href="https://www.jove.com/files/ftp_upload/65399/65399fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65399/65399fig10.jpg" /> 
Figure 10.&#160;OCR and LPR curves in response to test compounds. OCR and LPR by retina isolated from mice (1 retina/channel) in response to the presence or absence (control) of test compounds as indicated. Each curve is the average of 6 replicates from a single experiment (error bars are SE; p-values are calculated by performing paired t-tests comparing steady state values for each test agent with that of the previous test agent). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65399/65399fig11.jpg" /> 
Figure 11.&#160;OCR curves. OCR by RPE-choroid-sclera and retina isolated from mice (1 retina or 2 RPE-choroid-sclera/channel) measured in parallel in response to test compounds as indicated. Data is the average of replicates from a single experiment (n = 2 and 4 for RPE-choroid-sclera and retina respectively; p-values are calculated by performing paired t-tests comparing steady state values for each test agent with that of the previous test agent). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65399/65399fig12.jpg" /> 
Figure 12.&#160;OCR and LPR curves from RPE cells. OCR and LPR from RPE cells attached to transwell membranes that were cut into strips and loaded into the perifusion chambers. Data is the average of replicates from a single experiment (n = 3, with 1.5 membranes/channel (360,000 cells/channel); p-values are calculated by performing paired t-tests comparing steady state values for each test agent with that of the previous test agent). <a href="https://www.jove.com/files/ftp_upload/65399/65399fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>TISSUE/CELL</td>
			<td>Amount/Channel</td>
			<td>FLOW RATE: mL/min</td>
		</tr>
		<tr>
			<td>Retina (mouse)</td>
			<td>1</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>RPE-choroid-sclera (mouse)</td>
			<td>2</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>RPE Cells on Transwell Membranes</td>
			<td>360,000 Cells (4 x 1/3 filter strips)</td>
			<td>0.016</td>
		</tr>
	</tbody>
</table>
Table 1. Recommended operating specifications for different tissue.
Supplementary Figure 1. Graphical representation of experimental design. Timing and composition of exposure to test compounds, and timing of fraction collection. Concentration increment (Conc Inc) is the change in concentration to be implemented. <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2. User interface at startup. UI of the startup window of the O2 detection software that monitors the O2 in the tissue chambers inserted into the PCM. <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3. User interface for experiment settings. UI for entering experimental information (left) and selecting times for collection of outflow fractions (right). <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig3.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4. User interface of the injection page. Injection page which calculates injection volumes based on desired concentrations of test compound and volume left in the MRM. <a href="https://www.jove.com/files/ftp_upload/65399/Supp Fig4.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Methods for tissue sample preparation. <a href="https://www.jove.com/files/ftp_upload/65399/supplementary file 1_RE.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment at JoVE.com

Author Spotlight: Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell ...

maintaining-assessing-various-tissue-cell-types-eye-using-novel

Research

JoVE Journal

Bioengineering

1.2K Views.  EnTox Sciences, Inc. De meest gebruikte apparaten voor het meten van het zuurstofverbruik ondersteunen niet de gelijktijdige verzameling van uitstroomfracties. Het is van cruciaal belang om ook de uitstroom te testen om het metabolisme te correleren met de weefselfunctie. Door beide door technologie te meten, kunnen cellulaire mechanismen die weefselfunctie en ziekte mediëren, beter worden beoordeeld.De belangrijkste uitdaging bij het meten van het zuurstofverbruik en levend weefsel is het leveren van vol...

Video: Auteur in de schijnwerpers: Bevordering van oogfysiologisch onderzoek via een meerkanaals flowcultuur voor optimaal weefselonderhoud en real-time beoordeling 

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Medicine

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

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