Probe and system for measuring transepithelial resistance and transepithelial potential difference

Abstract

A probe (30) for a transepithelial resistance and transepithelial potential difference instrument (10) may include a temperature sensor configured to record a temperature in a well of a cell culture plate while collecting TEER or TEPD measurements. The temperature sensor may be surface mounted on one of the pins (36) of the probe, may be disposed within a tube disposed proximate to one of the pins of the probe, or, when the probe pin is tubular, may be disposed within a conduit of one of the pins. The top pins and the substrate outside pins may be formed as a printed circuit board assembly in which the sensing element is subjected to an electroless nickel plating and gold leaching process so as to be coated with a nickel layer followed by a gold layer. The top pin and the base outside pin may also be formed of 304 stainless steel, and may be tubular.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 503,028, filed May 18, 2023, entitled "Probe and System for Measuring Transepithelial Electrophysial Resistance and Transepithelial Potential Difference," the disclosure of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0002] This disclosure relates primarily to electrode probes and EVOM voltmeters, which are particularly used for measuring transepithelial resistance and transepithelial potential difference. Background Technology

[0003] Transepithelial resistance (TEER) is a commonly used non-destructive method for measuring the fusion and barrier integrity of cell monolayers. In TEER measurement, an alternating current signal (typically a 12.5 Hz square wave) of known current is applied across both sides of the cell monolayer. The voltage induced by this signal is conducted from the solution on either side of the cell monolayer. Since the voltage of this signal is measured and the current is known, Ohm's law (voltage = current * resistance) can be used to calculate the resistance of the monolayer. This technique is used during cell growth to assess the fusion progress of cell monolayers and to evaluate the final state of cells at a given experimental stage.

[0004] Transepithelial potential difference (TEPD) is a technique for measuring the potential difference (i.e., voltage) emitted by a cell monolayer. TEPD and TEER measurements use the same equipment. The voltage generated by the cell itself is conducted directly from the solution on either side of the cell monolayer, rather than by applying a known current to the system. This technique is used less frequently than TEER and is only applicable to certain cell types that generate potential differences.

[0005] There are generally three categories of devices / systems for measuring TEER / TEPD. These systems include: (a) single-up instruments and probe systems; (b) single-up instruments and cup systems; and (c) automated high-throughput systems. This invention relates to single-up instruments and probe systems, and indirectly to automated high-throughput systems.

[0006] Generally, single-function instrument and probe systems consist of two parts: (a) an instrument that reads the measurement and may generate an electrical signal; and (b) a probe that docks with the cell culture apparatus and transmits the electrical signal to and from the instrument. These systems are capable of measuring one experiment at a time (i.e., measuring the membrane across one well of a multi-well plate).

[0007] Conventional probes vary in overall shape but are composed of the same core features. Most conventional probes have two leads (a top lead and a base-outer lead). When in use, the top lead is located inside the hole insert in the plate, while the base-outer lead is located outside the hole insert but inside the hole itself. Furthermore, each conventional probe typically has four sensor elements: two elements are used to detect voltages induced or generated by the experiment, and two elements are used to apply a signal to the experiment for TEER measurement. Additionally, the surface of the sensing elements on conventional probes can be silver (as a coating or substrate). When in use, customers / users expect to immerse the probe in sodium hypochlorite or hydrochloric acid to convert the surface to silver / silver chloride (Ag / AgCl). Customers / users must perform this process periodically so that the surface can be recoated when removing the silver chloride.

[0008] It is well known that TEER measurements are temperature-dependent. However, currently, traditional TEER measurement systems do not integrate temperature measurement or control into their platforms. While high-end automated high-throughput systems may consider temperature, this is typically achieved by controlling the temperature of the environment in which TEER measurements are performed.

[0009] Automated high-throughput systems are dedicated platforms, typically incorporating chambers with environmental controls and custom-designed consumables for use within their platform. Some automated high-throughput systems can utilize existing orifice plates / plugs and employ multi-functional probes for measurement. However, all high-throughput systems currently on the market are complex, standalone systems. Currently, existing add-ons configured to convert single-function TEER platforms into high-throughput automated measurement systems are not available on conventional single-function TEER platforms.

[0010] Therefore, it is preferable to integrate a temperature measurement system (e.g., a temperature sensor, a thermistor, etc.) into at least one pin of the probe to allow TEER measurements to be performed in parallel with temperature measurements, thereby normalizing the measurement results. It is also desirable to formulate the sensing surface of the probe with materials other than silver / silver chloride to eliminate the need for periodic probe retreatment using sodium hypochlorite or hydrochloric acid. Furthermore, it is desirable to integrate networking capabilities into the TEER system to integrate the TEER system and TEER readings with cloud-based analysis software. These and other objectives are achieved through the embodiments disclosed herein. Summary of the Invention

[0011] This disclosure relates to a probe for transepithelial resistance and transepithelial potential difference (TEER) instruments, the probe including a temperature sensor configured to record the temperature in the wells of a cell culture plate while collecting TEER or TEPD measurements. The temperature sensor may be surface-mounted on one pin of the probe, disposed within a tube located near one of the probe pins, or, if the probe pin is tubular, disposed within a conduit of one of the pins. The top pin and the outer base pin may be formed as a printed circuit board assembly, wherein the sensing element undergoes a chemical nickel-immersion gold plating process to coat the sensing element with a nickel layer followed by a gold layer. The top pin and the outer base pin may also be formed of 304 stainless steel and may be tubular.

[0012] According to one embodiment, a probe for a transepithelial resistance and transepithelial potential difference instrument may include an upper housing, an electrode body coupled to the upper housing, a top pin coupled to the electrode body, a base outer pin coupled to the electrode body, and a temperature sensor disposed near the top pin or the base outer pin.

[0013] In some cases, the temperature sensor may be surface-mounted on a top pin, and the temperature sensor may be coated with an electrically insulating coating. In some other cases, the temperature sensor may be an encapsulated thermistor or thermocouple. In some other cases, the top pin may have an upper end, an opposite lower end, and at least one sensing element disposed near the lower end. In some other cases, the temperature sensor may be disposed close to at least one sensing element.

[0014] Furthermore, in some cases, the probe may also include a tube disposed near the tip pin, wherein the tube may have an upper end and a lower end, with the lower end of the tube disposed near the lower end of the tip pin. Additionally, a temperature sensor may be disposed within the lower end of the tube. Further still, the tube may be a stainless steel tube. In some other cases, the tube may be coupled to an electrode body; in other cases, the tube may be coupled to a tip pin. Furthermore, the tip pin may include a channel, and the tube may be disposed within the channel of the tip pin.

[0015] In some other cases, the instrument may include a network interface unit configured to send measurements acquired via probes to an analysis server over a network.

[0016] In another embodiment, the probe for the transepithelial resistor and transepithelial potential difference instrument may include an upper housing, an electrode body coupled to the upper housing, a top pin coupled to the electrode body, and a base outer pin coupled to the electrode body, wherein the top pin and the base outer pin are printed circuit board assemblies.

[0017] In some cases, the top pin may have an upper end, an opposite lower end, and at least one copper sensing element disposed near the lower end. In other cases, the outer pin may also have an upper end, an opposite lower end, and at least one copper sensing element disposed near the lower end. In still other cases, at least one copper sensing element of the top pin and at least one copper sensing element of the outer pin may be coated with nickel and gold layers via an electroless nickel-immersion gold plating process. In yet another case, the probe may include a temperature sensor disposed near the top pin or the outer pin.

[0018] In some other cases, the instrument may include a network interface unit configured to send measurements acquired via probes to an analysis server over a network.

[0019] In another embodiment, the probe for the transepithelial resistance and transepithelial potential difference instrument may include an upper housing, an electrode body coupled to the upper housing, a tubular tip pin coupled to the electrode body, and a tubular base outer pin coupled to the electrode body, wherein the tubular tip pin and the tubular base outer pin are constructed of an electrochemically inert and stable material.

[0020] In some cases, the tubular tip lead and the tubular base outer lead may be made of stainless steel. In other cases, the tubular tip lead and the tubular base outer lead may be made of lemon-passivated 304 stainless steel. Furthermore, the tubular tip lead may have an upper end, an opposite lower end, and a conduit extending from the upper end to the lower end. Additionally, in some further cases, a temperature sensor may be disposed near the lower end within the conduit of the tubular tip lead.

[0021] In some other cases, the instrument may include a network interface unit configured to send measurements acquired via probes to an analysis server over a network.

[0022] Other systems, apparatuses, devices, methods, features, and advantages will become apparent to those skilled in the art upon review of the following figures and detailed description. All such additional systems, apparatuses, arrangements, mechanisms, components, devices, methods, features, and advantages are included in this specification and are within the scope of the claimed subject matter. Attached Figure Description

[0023] A better understanding of the devices, systems, components, apparatuses, and parts presented herein can be achieved by referring to the following figures and descriptions. It should be understood that some elements in the figures may not be drawn to scale, and the emphasis has been placed on illustrating the principles disclosed herein. In the figures, the same reference numerals are used throughout different views to represent corresponding parts / steps.

[0024] Figure 1A perspective view of an exemplary embodiment of a single-function TEER instrument including a probe according to the present invention is shown.

[0025] Figure 2A A 24-well cell culture plate is shown, configured to support cell growth and... Figure 1 Used together with the single-function TEER instrument shown.

[0026] Figure 2B A 96-well cell culture plate is shown, configured to support cell growth and... Figure 1 Used together with the single-function TEER instrument shown.

[0027] Figure 3 This shows the probe of the disposable TEER instrument being inserted into Figure 1 , Figure 2A or Figure 2B A schematic diagram showing one of the wells in a cell culture plate for measuring transepithelial resistance.

[0028] Figures 4A-4C A perspective view of a probe according to a first embodiment of a disposable TEER instrument is shown, wherein a temperature sensor is integrated in one pin of the probe, and the pin of the probe is a printed circuit board assembly.

[0029] Figures 5A-5D A perspective view of a probe according to a second embodiment of a disposable TEER instrument is shown, wherein a temperature sensor is disposed in a tube near one of the probe's pins, and the probe's pins are printed circuit board assemblies.

[0030] Figure 5E It shows Figures 5A-5D The diagram shows a cross-sectional view of the probe, where the cross-section is along... Figure 5B The line 5E-5E in the middle is cut off.

[0031] Figures 6A-6D A perspective view of a probe according to a third embodiment of a disposable TEER instrument is shown, wherein the probe pins are tubular, and a temperature sensor is disposed within the tube of one pin of the probe.

[0032] Figure 6E It shows Figure 6C and Figure 6D The diagram shows a cross-sectional view of the probe, where the cross-section is along... Figure 6C The line BB in the middle is cut off.

[0033] Figure 7 The invention illustrates a method for constructing Figures 4A-4C and Figures 5A-5E The flowchart shows the method for attaching the probe pins.

[0034] Figure 8 A block diagram of a network environment according to an exemplary embodiment is shown, in which an embodiment of a single-function TEER instrument communicates with a cloud analytics tool.

[0035] Figure 9 A block diagram of a controller for a single-function TEER instrument according to one embodiment is shown, the controller being configured to perform the techniques described herein.

[0036] Figures 10A-10C An exemplary embodiment of a graphical user interface for a cloud-based analytics tool according to the present invention is shown, the analytics tool utilizing data acquired from a single-function TEER instrument. Detailed Implementation

[0037] In the following detailed description, reference is made to the accompanying drawings, which form a part of this detailed description, wherein like numerals denote like parts, and practical embodiments are illustrated in the drawings by way of illustration. It will be understood that other embodiments may be used, and structural or logical changes may be made without departing from the scope of this disclosure. Therefore, the following detailed description should not be considered limiting, and the scope of the embodiments is defined by the appended claims and their equivalents.

[0038] The aspects of this disclosure are disclosed in the description herein. Alternative embodiments and equivalents of this disclosure are conceivable without departing from the spirit or scope thereof. It should be noted that any discussion herein with reference to “one embodiment,” “an embodiment,” and “an exemplary embodiment,” etc., indicates that the described embodiments may include specific features, structures, or characteristics, and that such specific features, structures, or characteristics may not necessarily be included in every embodiment. Furthermore, references to the foregoing do not necessarily include references to the same embodiment. Finally, whether explicitly described or not, those skilled in the art will readily understand that each specific feature, structure, or characteristic of a given embodiment can be used in conjunction with features, structures, and characteristics of any other embodiment discussed herein.

[0039] Various operations can be described sequentially as a plurality of discrete actions or operations in a manner most conducive to understanding the claimed subject matter. However, the order of description should not be construed as implying that these operations necessarily depend on the order. In particular, these operations may not be performed in the order presented. The described operations may be performed in an order different from the described embodiments. In additional embodiments, various additional operations may be performed and / or the described operations may be omitted.

[0040] For the purposes of this disclosure, the phrase "A and / or B" means (A), (B), or (A and B). For the purposes of this disclosure, the phrase "A, B, and / or C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0041] The terms “comprising,” “including,” “having,” etc., as used in conjunction with the embodiments of this disclosure are synonyms.

[0042] Turning Figure 1 The illustration shows an exemplary embodiment of a voltmeter or single-function TEER system (hereinafter referred to as "TEER system") 10, configured to measure transepithelial resistance (hereinafter referred to as "TEER") in cell culture wells that may contain cells. The TEER system may include a measuring instrument 20 and probes 30 connected to the measuring instrument 20, wherein the probes 30 may be configured to be inserted into cell culture wells 42 of a cell culture plate 40, such as... Figure 1 As shown. As explained in further detail below, probe 30 may include electrode body 32, top pin 34, and substrate outer pin 36. Although Figure 1 The cell culture plate 40 shown includes 6 wells, but Figure 2A and Figure 2B Cell culture plates 50 and 60 of other embodiments are shown. Figure 2A The cell culture plate 50 shown is a 24-well plate (i.e., a cell culture plate with 24 wells 52), while Figure 2B The cell culture plate 60 shown is a 96-well plate (i.e., a cell culture plate with 96 wells 62).

[0043] Figure 3 A schematic embodiment of probe 20 for measuring the TEER of cells 70 in wells 42, 52, and 62 of cell culture plates 40, 50, and 60 is shown. As further illustrated, a suspension insert 44 is placed within the wells 42, 52, and 62 of the cell culture plates 40, 50, and 60, wherein the bottom surface 46 of the suspension insert 44 comprises a semi-permeable membrane on which cells 70 grow. When probe 20 is inserted into wells 42, 52, and 62, the top pin 34 is positioned within the suspension insert 44 (i.e., in the upper compartment of wells 42, 52, and 62), while the base outer pin 36 is positioned within wells 42, 52, and 62, but outside the suspension insert 44 (i.e., in the lower compartment of wells 42, 52, and 62). An alternating current voltage can be applied between the pins 34 and 36 of probe 30, where the resistance of the cell barrier tissue is measured / calculated by measuring instrument 10. Resistance, or TEER, is a quantitative parameter that measures the integrity of a cell monolayer through its ionic conductivity. Therefore, the TEER value increases with rapid cell proliferation and decreases when the barrier is damaged.

[0044] Go to Figures 4A-4C The diagram shows multiple perspective views of a probe 100 according to a first embodiment of the TEER system 10 of the present invention. The probe 100 can be used to measure the TEER of wells 52 of a 24-well cell culture plate 50. In other words, the probe 100 can be a 24-well probe. More specifically, the probe 100 includes a top housing 110 having a top end 112 and an opposite bottom end 114, wherein the top housing 110 defines an interior cavity 116 accessible via a top opening 117 in the top end 112 and a bottom opening 118 in the bottom end 114. The size of the top opening 117 may be smaller than the size of the bottom opening 118, and a cable 119 may be disposed within the top opening 117 such that the cable 119 extends from the top end 112 of the top housing 110.

[0045] Continue to refer to Figures 4A-4C The probe 100 also includes an electrode body 120 having a top end 122, an opposite bottom end 124, and a threaded sidewall 126 extending from the top end 122 to the bottom end 124. As further shown, the bottom end 114 of the top housing 110 may be threaded onto the top end 122 of the electrode body 120 such that the bottom end 114 of the top housing 110 at least partially surrounds the top end 122 of the electrode body 120 (and such that the electrode body 120 seals the interior 116 of the top housing 110 at the bottom opening 118). An electrode depth controller 130 may be threaded onto the bottom end 124 of the electrode body 120. The top end 132 of the electrode depth controller 130 may face or abut the bottom end 114 of the top housing 110 of the probe 100. The electrode depth controller 130 can be threaded onto the electrode body 120 and positioned in any location suitable for operating the probe 100, including but not limited to: the bottom end 134 of the electrode depth controller 130 being aligned with the bottom end 124 of the electrode body 120; the bottom end 134 of the electrode depth sensor 130 being positioned above the bottom end 124 of the electrode body 120; or the bottom end 134 of the electrode depth controller 130 being positioned below the bottom end 124 of the electrode body 120. Figure 4A In the illustrated embodiment, the electrode depth controller 130 may be transparent, while in other embodiments, the electrode depth controller 130 may be translucent or opaque.

[0046] Extending through the electrode body 120 can be a top pin 140 and a base outer pin 150. As shown, and because it is designed to be positioned within the upper compartment of the hole 52 of the cell culture plate 50, the length of the top pin 140 can be shorter than that of the base outer pin 150. The two pins 140 and 150 can each include upper ends 141 and 151 located within the cavity 116 of the top housing 110, respectively. Figure 4B(Best shown) and lower ends 142, 152. Top pin 140 may extend from the center of bottom end 124 of electrode body 120 such that top pin 140 and electrode body 120 are coaxial with each other. Substrate outer pin 150 may extend from bottom end of electrode body 120 near threaded sidewall 126 of electrode body 120.

[0047] As further shown, both the top pin 140 and the outer pin 150 of the substrate can be formed as a printed circuit board assembly (hereinafter referred to as "PCBA"), wherein copper or sensing elements 144, 154 are disposed on pins 140, 150, respectively, near the lower ends 142, 152. Furthermore, sensing elements 144, 154 can be disposed on the outer and inner sides of pins 140, 150. According to one embodiment, sensing elements 144, 154 can be formed as exposed copper pads, which are coated with nickel and subsequently with gold using an electroless nickel immersion gold (hereinafter referred to as "ENIG") PCB pad processing process. In another embodiment, sensing elements 144, 154 can be formed as exposed copper pads, which are coated with nickel and gold using an electrolytic nickel / gold (hereinafter referred to as "hard gold") PCB pad processing process, resulting in a hard gold plating layer on sensing elements 144, 154. Both pad processing techniques result in at least the surfaces of the sensing elements 144 and 154 being gold surfaces. Furthermore, processing the sensing elements 144 and 154 using either pad processing technique provides a flat and uniform surface, resulting in highly corrosion-resistant and oxidation-resistant sensing elements 144 and 154.

[0048] like Figures 4A-4C As further shown, probe 100 may include a temperature sensor or thermistor 160, which is integrated (e.g., surface-mounted) onto one of pins 140 and 150, respectively, near the lower ends 142 and 152 of pins 140 and 150. In the illustrated embodiment, temperature sensor 160 is disposed on the top pin 140 of probe 100, and near an inductive element 144 disposed on the outer side of the top pin 140. Furthermore, temperature sensor 160 may be conformally coated to isolate temperature sensor 160 from the experimental electrical circuit under test. Although Figures 4A-4C The image shows a surface-mount thermistor 160, but the temperature sensor can be any other type of temperature sensor, including but not limited to encapsulated thermistors, thermocouples, or any other temperature sensing element.

[0049] Go to Figures 5A-5E The diagram shows multiple perspective views and one cross-sectional view of the probe 200 according to a second embodiment of the TEER system 10 of the present invention. Figure 5ESimilar to probe 100 of the first embodiment, probe 200 of the second embodiment may be a 24-well probe designed for measuring the TEER of wells 52 in a 24-well cell culture plate 50. Similar to probe 100 of the first embodiment, probe 200 includes a top housing 210 having a top end 212 and an opposite bottom end 214, wherein the top housing 210 defines an interior cavity 216 accessible via a top opening 217 in the top end 212 and a bottom opening 218 in the bottom end 214. The size of the top opening 217 may be smaller than the size of the bottom opening 218, and a cable 219 may be disposed within the top opening 217 such that the cable 219 extends from the top end 212 of the top housing 210.

[0050] Continue to refer to Figures 5A-5E The probe 200 also includes an electrode body 220 having a top end 222, an opposite bottom end 224, and threaded sidewalls 226 extending from the top end 222 to the bottom end 224. As further shown, the bottom end 214 of the top housing 210 can be threadedly connected to the top end 222 of the electrode body 220 such that the bottom end 214 of the top housing 210 at least partially surrounds the top end 222 of the electrode body 220 (and such that the electrode body 220 seals the inner cavity 216 of the top housing 210 at the bottom opening 218). Figure 5E As shown in the best embodiment, the grounding element 228 can be connected to (i.e., threaded / screwed to, etc.) the top end 222 of the electrode body 220.

[0051] like Figures 5A-5E As further shown, and similar to the probe 100 of the first embodiment, the probe 100 of the second embodiment may include an electrode depth controller 230, which may be threadedly connected to the bottom end 224 of the electrode body 220. The top end 232 of the electrode depth controller 230 may face or abut the bottom end 214 of the top housing 210 of the probe 200. The electrode depth controller 230 may be threadedly connected to the electrode body 220 in any position suitable for operating the probe 200, including but not limited to, the bottom end 234 of the electrode depth controller 230 being aligned with the bottom end 224 of the electrode body 220, the bottom end 234 of the electrode depth controller 230 being positioned above the bottom end 224 of the electrode body 220, or the bottom end 234 of the electrode depth controller 230 being positioned below the bottom end 224 of the electrode body 220. The electrode depth controller 230 may be transparent, translucent, or opaque, and its outer wall surface may be knurled.

[0052] Extending through the electrode body 220 can be a top pin 240 and a base outer pin 250. For example... Figure 5C and Figure 5EAs best shown, and because it is designed to be positioned within the upper compartment of the well 52 of the cell culture plate 50, the length of the top pin 240 can be shorter than that of the outer pin 250. The two pins 240 and 250 may each include lower ends 242 and 252 and upper ends 244 and 254, wherein the upper ends 244 and 255 may be positioned within the inner cavity 216 of the top housing 210 (e.g., Figure 5E (As best shown in the diagram). The top pin 240 may extend from the center of the bottom end 224 of the electrode body 220, such that the top pin 240 and the electrode body 220 are coaxial with each other. The outer base pin 250 may extend from the bottom end 224 of the electrode body 220 near the threaded sidewall 226 of the electrode body 220.

[0053] As further shown, and similar to the probe 100 of the first embodiment, the top pin 240 and the outer pin 250 of the probe 200 of the second embodiment can both be formed as a PCBA, wherein copper or sensing elements 246, 256 are respectively disposed on the pins 240, 250 near the lower ends 242, 252. Furthermore, the sensing elements 246, 256 can be disposed on the outer and inner sides of the pins 240, 250. Similar to the sensing elements 144, 154 of the probe 100 of the first embodiment, the sensing elements 246, 256 can be formed as exposed copper pads, which are coated with nickel followed by gold using an ENIG PCB pad processing process or a hard gold PCB pad processing process, such that the surfaces of the pins 240, 250 have a gold surface at least at the locations of the sensing elements 246, 256. Furthermore, processing the inductive elements 246 and 256 using any of the pad processing techniques provides a flat and uniform surface for the inductive elements 144 and 154, which results in the inductive elements 246 and 256 being highly corrosion-resistant and oxidation-resistant.

[0054] The main difference between the probe 200 of the second embodiment and the probe 100 of the first embodiment is that it integrates a temperature sensor / thermometer 260. Figures 5A-5EIn the illustrated embodiment, the temperature sensor 260 may be disposed within a tube 270 that can be coupled to the top pin 240, rather than being disposed on the surface of the top pin 240. The temperature sensor 260 can be any type of temperature sensor, including but not limited to surface-mount thermistors, in-wall thermistors, thermocouples, or any other temperature sensing element. More specifically, the tube 270 may include a top pin 272, an opposite lower pin 274, and a channel or conduit 276 extending from the top pin 272 through the tube 270 to the lower pin 274. In some embodiments, the tube 270 may be a stainless steel tube. Furthermore, the temperature sensor 260 may be encapsulated within the conduit 276 of the tube 270 near the lower pin 274. The tube 270 may be coupled to the top pin 240 and / or the electrode body 220 via adhesive, glue, glue, epoxy resin, or any known connection mechanism. Figures 5A-5E In the illustrated embodiment, the top pin 240 may include a recess / cutout / channel 248, wherein a tube 270 may be coupled to the top pin 240 such that the tube 270 is at least partially disposed within the channel 248, and such that the lower end 274 of the tube 270 and the temperature sensor 260 are disposed near the sensing element 246. In other embodiments, the top pin 240 may not include any channel 248, and the tube 270 having the temperature sensor 260 may be coupled to or disposed along either side or edge of the top pin 240. In some embodiments, the tube 270 may be a stainless steel tube, while in other embodiments, the tube 270 may be made of any other suitable material that electrically insulates the temperature sensor 260 but enables the temperature sensor 260 to detect the temperature within the well 52 of the cell culture plate 50.

[0055] refer to Figures 6A-6E The figures shown are multiple perspective views and one cross-sectional view of probe 300(A) of the third embodiment and probe 300(B) of the fourth embodiment of the TEER system 10 according to the present invention. Figure 6E Unlike probe 100 of the first embodiment and probe 200 of the second embodiment, probe 300(A) of the third embodiment and probe 300(B) of the fourth embodiment can be 96-well probes designed for measuring TEER in wells 62 of a 96-well cell culture plate 60.

[0056] like Figure 6A and Figure 6BAs best shown, the probe 300(A) of the third embodiment includes a top housing 310 having a top end 312 and an opposite bottom end 314, wherein the top housing 310 defines an inner cavity 316 accessible via a top opening 317 in the top end 312 and a bottom opening 318 in the bottom end 314 of the top housing 310. The size of the top opening 317 may be smaller than the size of the bottom opening 318, and a cable 319 may be disposed within the top opening 317 such that the cable 319 extends from the top end 312 of the top housing 310.

[0057] Continue to refer to Figure 6A and Figure 6B The probe 300(A) also includes an electrode body 320 having a top end 322 and an opposite bottom end 324. However, unlike the probes 100 and 200 of the previous two embodiments, the electrode body 320 of the probe 300(A) of the third embodiment includes a smooth sidewall 326 extending from the top end 322 to the bottom end 324, rather than a threaded sidewall. Figure 6B As best shown, the bottom end 314 of the top housing 310 can slide above the top end 322 of the electrode body 320, such that the bottom end 314 of the top housing 310 at least partially surrounds the top end 322 of the electrode body 320 (and such that the electrode body 320 seals the inner cavity 316 of the top housing 310 at the bottom opening 318). Figure 6B As best shown, a gasket 327 can be provided within the bottom opening 318 of the top housing 310 of the probe 300(A), the gasket 327 being used to create a frictional fit between the top housing 310 and the electrode body 320. Similarly... Figure 6B As shown, and similar to the probe 200 of the second embodiment, one or more grounding elements 328 may be connected to (i.e., threaded / screwed to, etc.) the top end 322 of the electrode body 320.

[0058] The probe 300(B) of the fourth embodiment may be structurally more similar to the probes 100 and 200 of the first two embodiments than the probe 300(A) of the third embodiment. The probe 300(B) of the fourth embodiment may include a top housing 310 having a top end 312 and an opposite bottom end 314, wherein the top housing 310 defines an inner cavity 316 accessible via a top opening 317 in the top end 312 and a bottom opening 318 in the bottom end 314. The size of the top opening 317 may be smaller than the size of the bottom opening 318, and a cable 319 may be disposed within the top opening 317 such that the cable 319 extends from the top end 312 of the top housing 310.

[0059] Continue to refer to Figure 6C-6EThe probe 300(B) also includes an electrode body 320 having a top end 322, an opposite bottom end 324, and a threaded sidewall 326 extending from the top end 322 to the bottom end 324. As further shown, the bottom end 314 of the top housing 310 can be threadedly connected to the top end 322 of the electrode body 320 such that the bottom end 314 of the top housing 310 at least partially surrounds the top end 322 of the electrode body 320 (and such that the electrode body 320 seals the inner cavity 316 of the top housing 310 at the bottom opening 318). Figure 6E As best shown in the diagram, and similar to the probe 200 of the second embodiment, the grounding element 328 can be coupled (i.e., threaded / screwed to, etc.) to the top end 322 of the electrode body 320.

[0060] like Figure 6C-6E As further shown, unlike the probe 300(A) of the third embodiment, and similar to the probe 100 of the first embodiment and the probe 200 of the second embodiment, the probe 300(B) of the fourth embodiment may include an electrode depth controller 330, which may be threadedly connected to the bottom end 324 of the electrode body 320. The top end 332 of the electrode depth controller 330 may face or abut the bottom end 314 of the top housing 310 of the probe 300(B). In addition, the electrode depth controller 330 may be threadedly connected to the electrode body 320 in any position suitable for operating the probe 300(B), including but not limited to, the bottom end 334 of the electrode depth controller 330 being aligned with the bottom end 324 of the electrode body 320, the bottom end 334 of the electrode depth controller 330 being positioned above the bottom end 324 of the electrode body 320, or the bottom end 334 of the electrode depth controller 330 being positioned below the bottom end 324 of the electrode body 320. The electrode depth controller 330 can be transparent, translucent, or opaque, and the outer wall surface can be knurled.

[0061] Figures 6A-6E Further illustration shows that probe 300(A) of the third embodiment and probe 300(B) of the fourth embodiment both have a tip lead 340 extending through the electrode body 320 and a base outer lead 350. Since it is designed to be positioned within the upper compartment of the well 62 of the cell culture plate 60, the length of the tip lead 340 can be shorter than that of the base outer lead 350. Figure 6CAs shown in the optimal configuration, the two pins 340 and 350 may each include lower ends 342 and 352 and upper ends 344 and 354, respectively, wherein the upper ends 344 and 352 may be disposed within the inner cavity 316 of the top housing 310. Similar to the probes 100 and 200 of other embodiments, the top pin 340 of the probes 300(A) of the third embodiment and 300(B) of the fourth embodiment may extend from the center of the bottom end 324 of the electrode body 320, such that the top pin 340 and the electrode body 320 are coaxial with each other. The outer substrate pin 350 may extend from the bottom end 324 of the electrode body 320 near the threaded sidewall 326 of the electrode body 320. However, unlike the probe 100 of the first embodiment and the probe 200 of the second embodiment, the top pin 340 and the outer substrate pin 350 of the probes 300(A) of the third embodiment and 300(B) of the fourth embodiment may be constructed as conduits, which are preferably, but not limited to, constructed of an electrochemically inert and stable material. In one exemplary embodiment, the top lead 340 and the outer base lead 350 are constructed from 304 stainless steel tubing. More preferably, the 304 stainless steel tubing of leads 340 and 350 may be lemon passivated, which allows the stainless steel to possess electrochemical inertness similar to that of gold. Figure 6E As best shown in the diagram, the representation of the top pin 340 and the base side pin 350 can also represent the probe 300(A) of the third embodiment and the probe 300(B) of the fourth embodiment. The top pin 340 may include a conduit 346 extending from the upper end 344 to the lower end 342, while the base side pin 350 may also include a conduit 356 extending from the top end 354 to the lower end 352.

[0062] The probe 300(A) of the third embodiment and the probe 300(B) of the fourth embodiment also include an integrated temperature sensor / thermometer 360. For example... Figure 6E As shown in the optimal configuration, the temperature sensor 360 may be disposed within the conduit 346 of the tubular top pin 340 near the lower end 342 of the top pin 34. The temperature sensor 360 may be any type of temperature sensor, including but not limited to surface-mount thermistors, in-package thermistors, thermocouples, or any other temperature sensing element.

[0063] Go to Figure 7The diagram shows a flowchart 400 illustrating the steps for configuring or manufacturing the PCBA top pins 140, 240 and substrate outer pins 150, 250 of the probe 100 of the first embodiment and the probe 200 of the second embodiment. At step 405, the PCBA may be formed into the desired shape of the top pins 140, 240 or the substrate outer pins 150, 250 of the TEER probes 100, 200. Next, at step 410, the formed PCBA is subsequently laminated with one or more copper sensing elements 144, 154, 246, 256 at positions near the lower ends 142, 152, 242, 252 of the PCBA pins 140, 150, 240, 250. At step 415, one or more paths may be etched from the copper sensing elements toward the upper ends 141, 151, 244, 254 of the PCBA pins 140, 150, 240, 250. As previously described, and as... Figure 7 As shown, PCBA pins 140, 150, 240, and 250 can be processed using either ENIG pad processing or hard gold pad processing. In step 420, when PCBA pins 140, 150, 240, and 250 are processed using ENIG pad processing, a nickel layer is plated onto the copper sensing elements 144, 154, 246, and 256 using a chemical plating surface treatment process. Furthermore, in step 425, a gold layer is plated onto the nickel layer using a chemical plating surface treatment process. Conversely, in step 430, when PCBA pins 140, 150, 240, and 250 are processed using hard gold pad processing, a nickel layer is plated onto the copper sensing elements 144, 154, 246, and 256 using an electroplating surface treatment process. Furthermore, in step 435, a gold layer is plated onto the nickel layer using an electroplating surface treatment process. Finally, at step 440, whether using ENIG pad processing or hard gold pad processing, PCBA pins 140, 150, 240, and 250 are processed, and then PCBA pins 140, 150, 240, and 250 are attached to the electrode bodies 120 and 220 of TEER probes 100 and 200.

[0064] refer to Figure 8The diagram depicts an exemplary network environment 500 in which the TEER instruments of the disclosed embodiments communicate with each other and / or with the TEER analysis server 530. As shown, the TEER instruments 10(1)-10(N) (collectively referred to as "TEER instruments 10") may each include network interface units 510(1)-510(N) and may be operated by a local user to collect TEER measurements and temperature measurements. The network interface (I / F) units 510(1)-510(N) may be, for example, an Ethernet card or other interface device that allows the TEER instruments 10 to communicate via a communication network 520. Network I / F units 510(1)-510(N) may include wired and / or wireless connectivity, enabling each TEER instrument 10 (e.g., TEER instrument 10(1), TEER instrument 10(2), TEER instrument 10(N), etc.) to be able to and configured to send and / or receive data via communication network 520, particularly to communicate with TEER analysis server 530. Communication network 520 may include one or more wide area networks (WANs) (e.g., the Internet) and one or more local area networks (LANs). Each TEER instrument 10 may be a wired or wireless communication device, which may include, but is not limited to, wired or wireless communication devices. Figure 1 The exemplary embodiments shown include laptops, tablets, smartphones, etc.

[0065] Figure 8 Further details are provided regarding a cell imaging device 540 and a computing device 550. The cell imaging device 540 may be a device configured to monitor cells using a microscope, measure cell fusion, measure cell counts, and / or measure morphology. The cell imaging device 540 may also include a network interface unit 542, which enables the cell imaging device 540 to send and / or receive data via a communication network 520, particularly communicating with a TEER analysis server 530. The computing device 550 may be any wired or wireless communication device capable of communicating via the communication network 520, such as, but not limited to, a desktop computer, laptop computer, tablet computer, smartphone, etc.

[0066] In the transmission direction, the TEER instrument 10 captures TEER measurements and / or temperature measurements via probes 100, 200, 300(A), and 300(B) as described herein, encodes the captured measurements into data packets, and transmits the data packets to the TEER analysis server 530, the computing device 550, and / or the cell imaging device 540. In some embodiments, the TEER instrument 10 may receive data packets from the TEER analysis server 530, other TEER instruments 10, the computing device 550, and / or the cell imaging device 540. Similarly, in the transmission direction, the cell imaging device 540 may capture microscope images, encode the images into data packets, and transmit the data packets to the TEER analysis server 530, the computing device 550, and / or the TEER instrument 10. In some embodiments, the cell imaging device 540 may also receive data packets from the TEER analysis server 530, the TEER instrument 10, the computing device 550, and / or other cell imaging devices.

[0067] Now for reference Figure 9 It depicts an exemplary block diagram of the controller 600 of the TEER analysis server 530, which is configured to perform techniques for analyzing and correlating measurements and images captured by the TEER instrument 10 and the cell imaging device 540. Many possible configurations exist for the controller 600. Figure 9 This is just one example. Controller 600 includes processor 610, network interface unit 620, and memory 630. Network interface (I / F) unit (NIU) 620 is, for example, an Ethernet card or other interface device that allows controller 600 to communicate via communication network 520. Network I / F unit 620 may include wired and / or wireless connectivity capabilities.

[0068] Processor 610 may include a collection of microcontrollers and / or microprocessors, for example, each microcontroller and microprocessor being configured to execute corresponding software instructions stored in memory 630. A portion of memory 630 (and the instructions therein) may be integrated with processor 610. In the transmission direction, processor 610 encodes TEER measurements and temperature measurements captured by TEER instrument 10, as well as microscopic data from cell imaging device 540, encoding the captured measurements and images into data packets, and transmitting the encoded data packets to communication network 520. In the receiving direction, processor 610 decodes measurement data packets and imaging data packets received from communication network 520, and sends the decoded data packets to software instructions.

[0069] Memory 630 may include read-only memory (ROM), random access memory (RAM), disk storage media, optical storage media, flash memory, electrical, optical, or other physical / tangible (e.g., non-transient) storage devices. Therefore, in general, memory 630 may include one or more computer-readable storage media (e.g., storage devices) that are software-coded containing computer-executable instructions, and which, when executed (by processor 610), can operate to perform the operations described herein. For example, memory 630 stores or encodes instructions for TEER analysis logic 640, which facilitates correlating TEER measurements with temperature measurements, normalizing TEER measurements, correlating TEER measurements with cell microscopy images, and generating graphical output that can utilize the normalized and correlated data. The TEER analysis logic 640 includes: a TEER measurement correlation module 642 configured to correlate TEER measurements collected via probes 100, 200, 300(A), and 300(B) with temperature measurements collected via probes 100, 200, 300(A), and 300(B); a TEER measurement normalization module 644 configured to normalize TEER measurements based on correlated temperature measurements; a TEER measurement and cell image correlation module 646 configured to correlate TEER measurements collected via probes 100, 200, 300(A), and 300(B) of the TEER instrument 10 with microscopic images collected via the cell imaging device 540; and a plotting module 648 configured to generate a plot using TEER measurements, temperature measurements, and / or microscopic images. Figures 10A-10C The graphic images shown are for analysis purposes.

[0070] In addition, memory 630 may store data 650 used and generated by logic / modules 640-648, including but not limited to: TEER instrument identity (i.e., which TEER device recorded the measurement), TEER measurement, temperature measurement and / or cell image.

[0071] refer to Figures 10A-10C And as previously described, an exemplary embodiment of a graphical image that can be generated by the TEER analysis logic 640 of the TEER analysis server 530 is shown. Figure 10A A first exemplary embodiment of a graphical user interface 700 (hereinafter referred to as "GUI") generated by the TEER analysis logic 640 of the TEER analysis server 530 is shown. The GUI 700 depicts the resistance area (ohms*cm) of three sample groups (i.e., A, B, C). 2—A line graph along the y-axis versus the number of days. GUI 700 allows users to view temperature-corrected data and provides the option to change the y-axis to represent resistance (ohms), resistance area (ohms*cm). 2 The GUI 700 provides the ability to measure temperature (degrees Celsius) and voltage (V). It also depicts a graphical image of a culture plate (e.g., a 24-well plate) from which measurements are collected, with each well labeled. The GUI 700 provides additional information, such as, but not limited to, whether the data is archived, the user who created the data, the date the data was created, the instrument ID on which the measurements were performed, the user who last modified the data, and the date that user modified the data. The GUI 700 also enables users to provide additional data related to the calculations, such as, but not limited to, the name of the culture plate from which measurements were obtained, the type of culture plate (e.g., a 24-well plate), the type of insert placed in the wells of the culture plate, the growth area of ​​the insert membrane, the volume per well, and the cell seeding date. Finally, the GUI 700 allows users to input their own notes into data-related notes, and has the ability to download data summaries, delete measurement data, and / or archive data.

[0072] Go to Figure 10B The image shows another exemplary GUI 710, which provides users with the ability to perform graphical analysis of data. While GUI 710 plots the measurements of the three sample groups as a line graph, it offers users the ability to plot the data as bar charts, line graphs (as selected), pie charts, and other graphical options. GUI 710 also allows users to select which data to represent on the y-axis and x-axis. In the example depicted, the user has selected to view the resistance as a function of hours (x-axis) (y-axis). Finally, GUI 710 allows users to analyze and filter data by device, category, item, cell line, and plate.

[0073] Go to Figure 10C The image shown is another exemplary GUI 720 for presenting graphical analysis of data. Figure 10C The GUI 720 depicted includes charts that depict various values ​​(in this example, resistance) of a series of wells (or just the culture plate itself) over several days (i.e., 7 days). Users can select to display three groups for comparison and compare them with buffer values ​​within the same time period. GUI 720 also allows users to expand and collapse multiple wells in a specific group, add or remove sample groups, and change the displayed values.

[0074] By switching from silver / silver chloride electrodes to a more robust and electrically inert surface material, the need for periodic reprocessing of probes 100, 200, 300(A), and 300(B) with sodium hypochlorite or hydrochloric acid is eliminated. Furthermore, integrating temperature sensors 160, 260, and 360 onto the top pins 140, 240, and 340 of probes 100, 200, 300(A), and 300(B), respectively, allows users of the TEER system 10 to correlate TEER measurements with temperature. This further allows users and / or the system to normalize TEER measurements based on their associated temperature measurements, improving the accuracy of TEER measurements and experimental results. Moreover, by integrating the TEER system with the ability to communicate via a network and cloud computing environment, the TEER system allows: easy transfer of data from the TEER instrument to a server and / or personal computer for recording and analysis; optimization of advanced data analysis and graphics for TEER measurements; the ability to apply temperature compensation to TEER data based on temperature sensor readings; and the ability to correlate TEER data with microscopic images of cells.

[0075] While the devices described herein have been detailed and illustrated with reference to specific embodiments thereof, they are not intended to be limited to the details shown, as it will be apparent that various modifications and structural variations can be made within the equivalent scope of the claims without departing from the scope of the invention. For example, the probes described herein can be modified to include any number of housings, components / bodies, pin sensing elements, temperature sensors, etc.

[0076] Furthermore, various features from one embodiment may be combined in another embodiment. That is, it is believed that the foregoing disclosure covers several different inventions with independent practical applicability. While each of these inventions has been disclosed in its preferred form, the specific embodiments disclosed and described herein should not be considered limiting, as many variations are possible. The subject matter of this invention includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, and / or characteristics disclosed herein. Therefore, it is apparent that the appended claims should be interpreted broadly and in a manner consistent with the scope of this disclosure as set forth in the appended claims.

[0077] It should also be understood that the terms used herein (e.g., “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “inner,” “outer,” “next,” “surface,” etc.) describe reference points only and do not limit the invention to any particular orientation or configuration. Furthermore, the term “exemplary” is used herein to describe examples or illustrations. Any embodiment described herein as exemplary should not be construed as a preferred or advantageous embodiment, but rather as an example or illustration of a possible embodiment of the invention. Furthermore, it should be understood that components of the biological treatment system described herein, manifold assemblies described herein, or portions thereof may be made of any suitable material or combination of materials, such as, but not limited to, thermoplastics, plastics, or metals (e.g., copper, bronze, aluminum, steel, etc.) and their derivatives and combinations thereof. Furthermore, it should be understood that the steps of the methods described herein may be performed in any order or in any suitable manner.

[0078] Finally, when used herein, the term “comprising” and its derivatives (e.g., “including”, etc.) should not be construed as having an exclusionary meaning; that is, these terms should not be interpreted as excluding the possibility that the described and defined content may include other elements, steps, etc. Similarly, if any description refers to an element “a” or “first” or its equivalent, such disclosure should be understood to include a combination of one or more such elements, thus neither requiring nor excluding two or more such elements. Meanwhile, when used herein, the term “approximately” and its family of terms (e.g., “close to”, etc.) should be understood to indicate a value very close to the value following the foregoing term. That is, deviations from the precise value should be accepted, as those skilled in the art will understand that such deviations from the indicated value are unavoidable due to reasons such as measurement inaccuracies. The same applies to terms such as “about,” “around,” “approximately,” and “substantially.”

Claims

1. A probe for use in transepithelial resistance and transepithelial potential difference instruments, the probe comprising: Upper shell; An electrode body, which is connected to the upper housing; Top pin, which is connected to the electrode body; An outer pin of the substrate is connected to the electrode body; and A temperature sensor is disposed near the top pin or the outer pin of the base.

2. The probe according to claim 1, wherein, The temperature sensor is surface-mounted on the top pin and coated with an electrically insulating coating.

3. The probe according to claim 1, wherein, The temperature sensor is an encapsulated thermistor or thermocouple.

4. The probe according to claim 1, wherein, The top pin has an upper end, an opposite lower end, and at least one sensing element disposed near the lower end.

5. The probe according to claim 4, wherein, The temperature sensor is positioned near the at least one sensing element.

6. The probe according to claim 4, wherein, The probe also includes: A tube positioned near the top pin, the tube having an upper end and a lower end, wherein the lower end of the tube is positioned near the lower end of the top pin.

7. The probe according to claim 6, wherein, The temperature sensor is located inside the lower end of the tube.

8. The probe according to claim 7, wherein, The tube is connected to the electrode body.

9. The probe according to claim 7, wherein, The tube is connected to the top pin.

10. The probe according to claim 9, wherein, The top pin also includes a channel, and the tube is disposed within the channel of the top pin.

11. The probe according to claim 1, wherein, The instrument includes a network interface unit configured to send measurements acquired via the probe to an analysis server over a network.

12. A probe for use in instruments measuring transepithelial resistance and transepithelial potential difference, wherein, The probe includes: Upper shell; An electrode body, which is connected to the upper housing; Top pins, which are connected to the electrode body; and The outer pin of the substrate is connected to the electrode body, wherein the top pin and the outer pin of the substrate are printed circuit board assemblies.

13. The probe according to claim 12, wherein, The top pin has an upper end, an opposite lower end, and at least one copper sensing element disposed near the lower end. The outer pin of the base has an upper end, an opposite lower end, and at least one copper sensing element disposed near the lower end. The at least one copper sensing element of the top pin and the at least one copper sensing element of the outer pin of the base are coated with nickel and gold layers by an electroless nickel-gold immersion process.

14. The probe according to claim 13, wherein, The instrument includes a network interface unit configured to send measurements acquired via the probe to an analysis server over a network.

15. The probe according to claim 12, wherein, The probe also includes: A temperature sensor is disposed near the top pin or the outer pin of the base.

16. A probe for use in transepithelial resistance and transepithelial potential difference instruments, the probe comprising: Upper shell; An electrode body, which is connected to the upper housing; A tubular tip pin, which is connected to the electrode body; and The tubular substrate outer pin is connected to the electrode body, wherein the tubular top pin and the tubular substrate outer pin are constructed of stainless steel.

17. The probe according to claim 16, wherein, The tubular tip pin and the tubular base outer pin are constructed of citric acid passivated 304 stainless steel.

18. The probe according to claim 16, wherein, The instrument includes a network interface unit configured to send measurements acquired via the probe to an analysis server over a network.

19. The probe according to claim 16, wherein, The tubular tip pin has an upper end, an opposite lower end, and a conduit extending from the upper end to the lower end.

20. The probe according to claim 19, wherein, The probe also includes: A temperature sensor is disposed near the lower end within the conduit of the tubular top pin.

Images