IND
`
`US 20190376014A1
`
`IN
`
`( 19 ) United States
`( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2019/0376014 A1
`( 43 ) Pub . Date :
`Dec. 12 , 2019
`EFIMOV et al .
`
`( 71 )
`
`( 54 ) APPARATUS AND METHODS FOR IN VITRO
`PRECLINICAL HUMAN TRIALS
`Applicants : Igor R. EFIMOV , Arlington , VA ( US ) ;
`Yun QIAO , Arlington , VA ( US ) ;
`Chaoyi KANG , Arlington , VA ( US ) ;
`Zhenyu LI , McLean , VA ( US ) ; Quan
`DONG , Fairfax , VA ( US ) ; Baichen LI ,
`Falls Church , VA ( US )
`( 72 ) Inventors : Igor R. EFIMOV , Arlington , VA ( US ) ;
`Yun QIAO , Arlington , VA ( US ) ;
`Chaoyi KANG , Arlington , VA ( US ) ;
`Zhenyu LI , McLean , VA ( US ) ; Quan
`DONG , Fairfax , VA ( US ) ; Baichen LI ,
`Falls Church , VA ( US )
`( 21 ) Appl . No .:
`16 / 479,297
`( 22 ) PCT Filed :
`Jan. 24 , 2018
`( 86 ) PCT No .:
`$ 371 ( c ) ( 1 ) ,
`( 2 ) Date :
`Jul . 19 , 2019
`Related U.S. Application Data
`( 60 ) Provisional application No. 62 / 450,412 , filed on Jan.
`25 , 2017 .
`
`PCT / US2018 / 015052
`
`Publication Classification
`
`AOIN 1/02
`C12M 1/00
`CI2N 5/071
`GOIN 33/50
`C12N 9/22
`C12N 15/11
`C12M 1/36
`GOIN 1/36
`( 52 ) U.S. CI .
`CPC
`
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`C12M 23/16 ( 2013.01 ) ; GOIN 2001/368
`( 2013.01 ) ; C12N 5/0657 ( 2013.01 ) ; C12M
`27/02 ( 2013.01 ) ; C12M 41/18 ( 2013.01 ) ;
`C12M 41/26 ( 2013.01 ) ; AOIN 1/0247
`( 2013.01 ) ; AOIN 1/0284 ( 2013.01 ) ; AOIN
`1/021 ( 2013.01 ) ; AOIN 1/0278 ( 2013.01 ) ;
`C12M 29/10 ( 2013.01 ) ; C12N 570697
`( 2013.01 ) ; GOIN 33/5014 ( 2013.01 ) ; GOIN
`33/5088 ( 2013.01 ) ; C12N 9/22 ( 2013.01 ) ;
`C12N 15/11 ( 2013.01 ) ; C12M 27/16
`( 2013.01 ) ; C12M 41/34 ( 2013.01 ) ; C12M
`41/06 ( 2013.01 ) ; C12M 41/48 ( 2013.01 ) ;
`GOIN 1/36 ( 2013.01 ) ; BOIL 2300/0819
`( 2013.01 ) ; BOIL 2200/10 ( 2013.01 ) ; BOIL
`2300/0663 ( 2013.01 ) ; BOIL 2200/143
`( 2013.01 ) ; BOIL 2300/12 ( 2013.01 ) ; BOIL
`2200/025 ( 2013.01 ) ; BOIL 2400/0487
`( 2013.01 ) ; BOIL 2300/18 ( 2013.01 ) ; BOIL
`2300/10 ( 2013.01 ) ; BOIL 2400/0415
`( 2013.01 ) ; BOIL 2200/185 ( 2013.01 ) ; BOIL
`2300/0645 ( 2013.01 ) ; BOIL 2300/027
`( 2013.01 ) ; C12N 2310/20 ( 2017.05 ) ; C12N
`2800/80 ( 2013.01 ) ; BOIL 3/502715 ( 2013.01 )
`( 57 )
`ABSTRACT
`Systems comprising a microfluidic device are provided for
`maintaining and analyzing tissue slices . Methods for main
`taining tissue slices in a microfluidic device are further
`provided
`
`( 51 ) Int . CI .
`C12M 3/06
`BOIL 3/00
`C12N 5/077
`C12M 1/06
`C12M 1/02
`C12M 1/34
`
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`
`Electronics
`
`1413
`
`1414
`
`1420
`
`-1410
`
`1400
`
`Temperature
`Acquisition
`Voltage Follower
`
`ECG Acquisition
`
`1417
`ADC
`
`DAC
`
`SPI
`
`Battery
`
`-1412
`
`1411
`
`Microcontroller
`( e.g. , Teensy 3.2 )
`
`Digital
`
`PWM
`
`Voltage Regulator
`
`Pump Controller
`
`1416
`
`1415
`
`Microfluidic culture chamber
`
`-1420
`
`Pheumatics
`
`1430
`
`Culture Chamber
`
`Sensing electrodes
`
`Pacing electrodes
`
`Temperature Sensor
`
`1422
`
`1423
`
`1427
`
`Pump ( s )
`( e.g. , Multichannel
`Low - power Pumps )
`
`1431
`
`1434
`
`Heater
`
`1435
`
`1433
`
`1432
`
`Gas Exchanger
`
`Filter
`( 5 um )
`
`Culture Medium
`Reservoir
`
`1436
`
`Pressure
`Regulator
`1439
`
`Portable
`Oxygen Tank
`
`APPLE 1063
`Apple v. AliveCor
`IPR2021-00970
`
`1
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 1 of 23
`
`US 2019/0376014 A1
`
`Day 0
`
`Day 2
`
`FIG . 1A
`
`Day 4
`
`65 6
`
`40
`
`20
`
`Oms
`
`40
`
`20
`
`Oms
`
`FIG . 1B
`
`Day 6
`
`90
`
`60
`
`30
`
`Oms
`
`65
`
`40
`
`20
`
`Oms
`
`FIG . 1C
`
`FIG . 10
`
`2
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 2 of 23
`
`US 2019/0376014 A1
`
`30
`
`25
`Conduction Velocity ( cm / s )
`20
`
`
`
`10
`
`5
`
`o
`
`15 Im
`
`Day 0
`
`Day 2
`
`Day 4
`
`Day 6
`
`FIG . 2
`
`3
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 3 of 23
`
`US 2019/0376014 A1
`
`300
`
`315A 335A
`315B
`
`Pump Controller ( e.g. , Servoflo MP6 )
`
`
`
`Pump Controller ( e.g. , Servoflo
`
`MP6 )
`
`311
`
`-312
`Battery
`
`310
`
`313
`
`Electronics
`
`..........
`
`PWM
`
`PWM
`
`Microcontroller ( e.g , Teensy
`
`3.2 )
`
`DAC
`
`SPI
`
`
`
`Voltage Follower ( e.g. , OP777 )
`
`
`
`ECG Acquisition ( e.g. , TI ADS1198 )
`
`314
`
`331B
`
`332
`
`331A
`
`diaphragm micropump )
`
`Pump
`( e.g. , Piezoelectric
`
`Medium Reservoir
`Culture
`
`diaphragm micropump )
`
`Pump
`( e.g. , Piezoelectric
`
`333
`
`Filter ( e.g. , 5 um )
`
`Air
`
`FIG . 3
`
`330
`
`Pneumatics
`
`320
`
`321
`
`322
`
`323
`
`
`
`
`
`Microfluidic culture chamber
`
`
`
`Sensing electrodes / sensors ( e.g. , Ag / AgCl )
`
`
`
`Pacing electrodes ( e.g. , Ptir )
`
`
`
`Culture Chamber ( e.g. , with
`
`PDMS )
`
`4
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 4 of 23
`
`US 2019/0376014 A1
`
`443
`
`444C
`
`422
`
`445
`
`444B
`
`a
`
`446
`
`421
`
`424
`
`420
`
`O
`
`444A
`
`444D
`
`442
`
`441
`
`En
`440 440 L
`
`423
`
`FIG . 4A
`
`5
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 5 of 23
`
`US 2019/0376014 A1
`
`443
`
`445
`
`444
`
`444
`
`440
`
`FIG . 4B
`
`421
`
`6
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 6 of 23
`
`US 2019/0376014 A1
`
`521A
`
`521B
`
`543
`
`521A
`
`543
`
`521B
`
`521A
`
`521B
`
`521D
`
`543
`
`540
`
`540
`
`FIG . 5A
`
`FIG . 5B
`
`540
`
`FIG . 5C
`
`521A
`
`540
`
`4
`
`FIG . 5D
`
`521C
`
`521C
`
`543
`
`521B
`
`521D
`
`7
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 7 of 23
`
`US 2019/0376014 A1
`
`600
`
`621
`
`643
`621
`2
`
`000 jo
`
`o
`
`640
`
`620
`
`1
`
`Sensing
`
`-612
`
`Power Supply
`
`-611
`
`Control Unit
`
`FIG . 6
`
`601A
`
`Reservoir 1
`
`Reservoir 2
`Reservoir 3
`
`601B
`
`601c
`
`Actuation
`
`bursuas
`
`8
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 8 of 23
`
`US 2019/0376014 A1
`
`ww
`
`1
`
`1
`
`Actuation
`
`Sensing
`
`Optical control
`
`Pharmacological and genetic
`intervention
`
`Enviornmental intervention
`( pH , temperature , Oz , etc. )
`
`Pneumatic control
`
`FIG . 7
`
`9
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 9 of 23
`
`US 2019/0376014 A1
`
`mmmonly
`
`60
`
`50
`
`40
`
`Time ( s )
`30
`
`20
`
`10
`
`o
`
`o
`
`( udq ) ?ley Je?H
`
`FIG . 8
`
`10
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 10 of 23
`
`US 2019/0376014 A1
`
`1 sec
`
`that the
`
`
`Without medium oxygenation
`they
`
`92 bpm
`
`1 sec
`
`7 bpm 75
`227 bpm
`
`FIG . 9
`
`
`
`
`
`With medium oxygenation
`
`11
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 11 of 23
`
`US 2019/0376014 A1
`
`1000
`
`1043
`
`1046
`
`1045
`
`FIG . 10
`
`1022
`
`1040
`
`1023
`
`1020
`
`12
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 12 of 23 US 2019/0376014 A1
`
`.
`
`30
`Conduction Velocity ( cm / s )
`20
`
`
`
`10
`
`Day 0 ( n = 8 ) Day 2 ( n = 8 ) Day 4 ( n = 5 ) Day 8 ( n = 2 )
`Day 6 ( n = 2 )
`
`FIG . 11
`
`Day 0
`- Day 21
`
`word
`
`250ms
`
`FIG . 12
`
`13
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 13 of 23
`
`US 2019/0376014 A1
`
`Day 0
`
`Day 21
`
`FIG . 13A
`
`65
`
`40
`
`20
`
`0
`
`500
`
`400
`
`300
`
`200
`
`FIG . 13B
`
`100
`
`50
`
`0
`
`500
`
`400
`
`300
`
`200
`
`FIG . 13C
`
`FIG . 13D
`
`14
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 14 of 23
`
`US 2019/0376014 A1
`
`1400
`
`1410
`
`Electronics
`
`1415
`
`1416
`
`Voltage Regulator
`
`Digital
`
`
`
`Pump Controller
`
`PWM
`
`Microcontroller ( e.g. , Teensy
`
`3.2 )
`
`DAC
`
`SPI
`
`
`
`Voltage Follower
`
`
`
`ECG Acquisition
`
`1411
`
`1412
`
`Battery
`
`ADC
`1417
`
`Temperature Acquisition
`
`1436
`
`1439
`
`Pressure Regulator
`
`Portable Oxygen
`Tank
`
`1431
`
`1434
`
`Pump ( s )
`
`
`Low - power Pumps )
`( e.g. , Multichannel
`
`Heater
`
`
`
`Gas Exchanger
`
`Medium Reservoir
`Culture
`
`1435
`
`1432
`
`1433
`
`Filter ( 5 pm )
`
`FIG . 14
`
`-1430
`
`Pheumatics
`
`1420
`
`
`
`
`
`Microfluidic culture chamber
`
`1422
`
`1423
`
`1427
`
`
`
`Culture Chamber
`
`
`
`Sensing electrodes
`
`
`
`Pacing electrodes
`
`
`
`Temperature Sensor
`
`-
`
`1414
`
`1420
`
`1413
`
`15
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 15 of 23
`
`US 2019/0376014 A1
`
`1500
`
`FIG . 15
`
`1521
`
`1510
`
`1535
`
`1560
`
`1536
`
`16
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 16 of 23
`
`US 2019/0376014 A1
`
`1614
`
`D
`
`0000000000000 "
`
`220
`
`OM
`ooooooooooooooo
`
`oo
`
`1615
`
`1611
`
`MD
`
`OM OM
`
`1612
`
`0000
`
`Olc0000
`
`FIG . 16A
`
`1611
`
`1640
`
`1600
`
`1634
`
`0000ogooo
`
`?
`
`ooooo0000000
`
`TUIT
`
`COOL 00
`
`Orgung
`
`1620
`
`1643
`
`FIG . 16B
`
`1620
`
`1622
`
`Uhr
`
`it
`
`1614
`
`1628
`1627
`
`1623
`FIG . 16C
`
`17
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 17 of 23
`
`US 2019/0376014 A1
`
`SWS
`
`ses
`
`V
`
`Time ( s )
`
`windows
`
`W ) apnidur buroed
`
`1711
`
`1721
`
`000
`
`ooooo ooogoo
`00000
`
`TIT
`
`000000
`
`ooo
`
`FIG . 18
`
`FIG . 17
`
`18
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 18 of 23 US 2019/0376014 A1
`
`2139
`
`4
`
`ü
`
`2031
`
`1935
`
`FIG . 21
`
`FIG . 20
`
`FIG . 19
`
`19
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 19 of 23
`
`US 2019/0376014 A1
`
`02
`
`2
`
`30
`
`( uw ) aw
`15
`FIG . 23
`
`XN
`
`X
`
`nalized
`
`
`
`level Normalized 02
`
`
`
`Heater Culture chamber
`
`*****
`
`Time ( min )
`
`FIG . 22
`
`Se
`
`44
`
`)
`
`( C
`
`Temperature
`
`20
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 20 of 23
`
`US 2019/0376014 A1
`
`50
`
`40
`
`30
`
`20
`
`10
`
`o
`
`Human Day 3
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Human Day 1
`
`40
`
`30
`
`20
`
`10
`
`a
`
`Human Day 0
`
`FIG . 24C
`
`FIG . 24B
`
`FIG . 24A
`
`21
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 21 of 23
`
`US 2019/0376014 A1
`
`16
`
`8 12 Time ( hr )
`
`FIG . 26
`
`4
`
`700
`)
`
`500
`
`( bpm
`HR Murine Atria
`
`100
`
`
`0
`
`
`
`Time ( s )
`
`0.2
`
`FIG . 27
`
`???
`
`1000
` ( mV ) Signal Amplitude
`
`
`
`
`Day 0 Day 1 Day 3
`
`MOM
`
`500ms
`
`FIG . 25
`
`22
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 22 of 23
`
`US 2019/0376014 A1
`
`Section 2
`
`Section 3
`
`X 104
`
`X 104
`
`X104
`
`X 104
`
`FIG . 28
`
`25
`
`5
`
`5
`
`Time ( s )
`
`Time ( s )
`
`Time ( s )
`
`belakaan
`
`Time ( s )
`
`2
`
`2
`
`memposing
`
`Johocu
`
`X104
`
`2.8
`
`BPM 22
`532
`
`2.65
`
`26
`
`800
`
`600 * 400
`
`2007 0 .
`
`2:55
`-200 400
`
`Second Pod
`
`w
`
`Chamber 3
`
`2.4000
`800 600 400 2002 0 -200
`
`) Amplitude ( mv
`
`
`05
`
`Chamber
`
`( 0 )
`
`Temp
`
`300 200 1001 0 100 % 200 % 300 4000
`
` Amplitude ( mm )
`
`Chambeni
`
`1500 1000
`
`
`
`0 500 10000
`
`
`
`) Amplitude ( MV
`
`
`Device log / 1215 mousellog : Jog Folder / Culture
`
`
`
`
`
`Culture Device Shared Load Log File WUsers / John / Box / Current Projects / Slice
`
`
`Plot ECG
`
`
`
`Peak Detection
`
`10 11 12 13 14 15 16 17 18 19 20 21
`ooo
`
`Section 1
`
`
`
`
`
`
`
`Flip Graph 3 5 100 Flip Graph 1 15 100 Flip Graph 2 .5 100
`
`
`
`Threshold Min Width
`
`
`
`Detect Peaks
`
`Section 4
`
`23
`
`

`

`Patent Application Publication
`
`Dec. 12 , 2019 Sheet 23 of 23
`
`US 2019/0376014 A1
`
`2900
`
`2943
`
`2940
`
`2920 ,
`
`2922
`
`2923
`
`2944
`
`2927
`
`FIG . 29B
`
`2950
`
`2911
`
`2931
`
`FIG . 29A
`
`2935
`
`2939
`
`2920
`
`24
`
`

`

`US 2019/0376014 A1
`
`1
`
`Dec. 12 , 2019
`
`APPARATUS AND METHODS FOR IN VITRO
`PRECLINICAL HUMAN TRIALS
`
`CROSS - REFERENCE TO RELATED
`APPLICATIONS
`This application claims the benefit of U.S. Provi
`[ 0001 ]
`sional Application No. 62 / 450,412 , filed Jan. 25 , 2017 ,
`which is incorporated herein by reference in its entirety .
`STATEMENT REGARDING
`FEDERALLY - SPONSORED RESEARCH AND
`DEVELOPMENT
`[ 0002 ] “ This invention was made with U.S. Government
`support under grants R01 HL114395 and R01 HL126802
`from the National Heart , Lung and Blood Institute of
`National Institutes of Health . “ The U.S. Government has
`certain rights in this invention .
`FIELD OF THE INVENTION
`[ 0003 ] The present subject matter relates , in general , to
`novel platforms and methods for the investigation of normal
`and pathological physiology , including human cardiac
`physiology , in vitro and the long - term organotypic culture of
`tissue , including human cardiac tissue , for in vitro pre
`clinical testing
`BACKGROUND OF THE INVENTION
`[ 0004 ] Physiologically and genetically accurate models of
`the human heart are indispensable for studying human
`cardiac physiology and for pre - clinical screening of candi
`date biological and drug therapies for their efficacy and / or
`toxicity . However , pre - clinical screening has been mostly
`limited to animal models and / or cell lines , which do not fully
`recapitulate human biology . Few studies have demonstrated
`long - term functional recordings from the human heart due to
`difficulties in obtaining and maintaining electrically viable
`tissue samples , and lack of reliable model systems of the
`human myocardium .
`[ 0005 ] Although crucial for fundamental biological dis
`covery , animal models often fail to predict human response
`to treatments due to inter - species genetic and physiological
`differences ( Hasenfuss , G. , Cardiovasc . Res . 39 ( 1 ) : 60-76
`( 1998 ) ; Mak , I. W. Y. et al . , Am . J. Transl . Res . 6 ( 2 ) : 114-8
`( 2014 ) ; Nerbonne , J. M. et al . , Circ . Res . 89 ( 11 ) : 944-56
`( 2001 ) ; FANTOM Consortium and the RIKEN PMI and
`CLST ( DGT ) et al . , Nature 507,462-70 ( 2014 ) , each of
`which is incorporated herein by reference in its entirety ) . In
`recent years , vibratome - cut human cardiac slices from donor
`and end - stage failing hearts have emerged as a promising
`model of the human heart for electrophysiological and
`pharmacological studies ( Brandenburger , M. et al . , Cardio
`vasc . Res . 93 ( 1 ) : 50-9 ( 2012 ) ; Camelliti , P. et al . , J. Mol .
`Cell . Cardiol . 51 ( 3 ) , 390-8 ( 2011 ) , each of which is incor
`porated herein by reference in its entirety ) . It has been
`demonstrated that human cardiac slices faithfully recapitu
`late tissue level human cardiac physiology , exhibiting nor
`mal conduction velocity ( CV ) , action potential duration
`( APD ) , intracellular calcium dynamics , heart rate depen
`dence of these parameters and their response to a- and
`B - adrenergic stimulation ( Kang , C. et al . , Sci . Rep . 6 : 28798
`( 2016 ) , which is incorporated herein by reference in its
`entirety ) . However , previous studies have only achieved
`short - term organotypic culture of human cardiac slices .
`
`[ 0006 ] Extensive efforts have been invested into develop
`ing an authentic model of the human heart . Human induced
`pluripotent stem cell derived cardiomyocytes ( hiPSC - CMS )
`are widely used in modeling diseases and drug screening
`( Chi , K. R. , Nat . Rev. Drug Discov . 12 ( 8 ) : 565-7 ( 2013 ) ;
`Itzhaki , I. et al . , Nature 471 ( 7337 ) : 225-9 ( 2011 ) , each of
`which is incorporated herein by reference in its entirety ) .
`However , the development of hiPSC - CMs with mature atrial
`or ventricular phenotype has been challenging so far ( Kara
`kikes , I. et al . , Circ . Res . 117 ( 1 ) : 80-8 ( 2015 ) ; Robertson , C.
`et al . , Stem Cells 31 ( 5 ) : 829-37 ( 2013 ) , each of which is
`incorporated herein by reference in its entirety ) .
`[ 0007 ] Another approach to study human cardiac cell
`biology involved isolated primary human cardiomyocytes ,
`which are functionally mature , but have limited experimen
`tal duration since they dedifferentiate in cell culture ( Bird , S.
`D. et al . , Cardiovasc . Res . 58 ( 2 ) : 423-34 ( 2003 ) ; Coppini , R.
`et al . , J. Vis . Exp . 86 : e51116 ( 2014 ) , each of which is
`incorporated herein by reference in
`its entirety ) . Different
`cardiomyocyte subpopulations can be obtained by altering
`the cell isolation process . However , they exhibit altered
`electrophysiology , i.e. action potential morphology , due to
`the lack of cell - cell coupling and membrane protein altera
`tions caused by the tissue digestion procedure . The cell
`isolation procedure is also time consuming and labor inten
`sive , thus limiting the use of isolated cardiomyocytes to
`low - throughput testing .
`[ 0008 ] Another approach is based on the human ventricu
`lar wedge preparations , which allow for studying CV , con
`duction heterogeneity , and arrhythmia susceptibility ( Gluk
`hov , A. V. et al . , Circulation 125 ( 15 ) : 1835-47 ( 2012 ) ;
`Glukhov , A. V. et al . , Circ . Res . 106 ( 5 ) : 981-91 ( 2010 ) ; Lou ,
`Q. et al . , Circulation 123 ( 17 ) : 1881-90 ( 2011 ) , each of which
`is incorporated herein by reference in its entirety ) . Due to the
`complexity and variability of the coronary system and the
`size constraint of the preparation , the ventricular wedge
`preparation is also severely limited in terms of throughput .
`Primary cells , cell lines , and tissue also have significantly
`different gene expression profiles , as reported by the func
`tional annotation of the Functional Annotation of the Mam
`malian Genome 5 ( FANTOM5 ) consortium ( FANTOM
`Consortium and the RIKEN PMI and CLST ( DGT ) et al . ,
`Nature 507,462-70 ( 2014 ) ) .
`[ 0009 ] Human cardiac slices faithfully replicate the organ
`level adult cardiac physiology because they retain the nor
`mal tissue architecture , multiple cell type environment , and
`extracellular matrix . ( Kang , C. et al . , Sci . Rep . 6 : 28798
`( 2016 ) ) . Therefore , human cardiac slices are advantageous
`over other in vitro and ex vivo model systems of cardiac
`physiology . For example , when compared with hiPSC - CMs ,
`human cardiac slices more faithfully replicate adult human
`cardiac electrophysiology with a mature myocyte pheno
`type , tissue structure and multicellular environment . When
`compared with isolated human myocytes , cardiac slices
`largely preserve the natural multicellular environment and
`coupling with the surrounding myocytes , helping to main
`tain the fully differentiated cellular and tissue phenotype .
`Maintenance of the native tissue context is especially impor
`tant for testing human gene therapy approaches . When
`compared with human ventricular wedge preparations , car
`diac slices do not require intact human ventricles and can be
`obtained from small biopsy samples . Most importantly ,
`human cardiac slices can be prepared at a thickness around
`the diffusion limit of oxygen . At the diffusion limit for
`
`25
`
`

`

`US 2019/0376014 A1
`
`2
`
`Dec. 12 , 2019
`
`oxygen , human cardiac slices can be cultured long - term for
`studying chronic drug treatment , gene expression regulation ,
`and genetic engineering ( Barclay , C. J. , J. Muscle Res . Cell
`Motil . 26 ( 4-5 ) : 225-35 ( 2005 ) ; Brandenburger , M. et al . ,
`Cardiovasc . Res . 93 ( 1 ) : 50-9 ( 2012 ) ; Bussek , A. et al . , J.
`Pharmacol . Toxicol . Methods 66 ( 2 ) : 145-51 ( 2012 ) ; Kang ,
`C. et al . , Sci . Rep . 6 : 28798 ( 2016 ) , each of which is
`incorporated herein by reference in its entirety ) . However ,
`there are significant drawbacks to previously disclosed stud
`ies utilizing human cardiac slices .
`[ 0010 ] Previous studies on the organotypic culture of
`ventricular slices obtained from adult mammalian hearts
`have been limited to about two days , which diminishes the
`usefulness of the preparation for testing the effects of
`chronic drug and gene therapies ( Brandenburger , M. et al . ,
`Cardiovasc . Res . 93 ( 1 ) : 50-9 ( 2012 ) ; Bussek , A. et al . , J.
`Pharmacol . Toxicol . Methods 66 ( 2 ) : 145-51 ( 2012 ) ; Kang ,
`C. et al . , Sci . Rep . 6 : 28798 ( 2016 ) ) . Lacking pacemaking
`abilities , slices collected from the ventricles of the heart
`undergo significant dedifferentiation , when cultured with
`conventional tissue culture techniques that lack electrical or
`mechanical stimulation and loading ( Brandenburger , M. et
`al . , Cardiovasc . Res . 93 ( 1 ) : 50-9 ( 2012 ) ; Kaneko , M. et al . ,
`J. Cell Sci . Ther . 3 ( 4 ) : e1000126 ( 2012 ) , each of which is
`incorporated herein by reference in its entirety ) . Human
`tissue and primary cells have been implemented in body
`on - a - chip systems designed for drug testing ( Esch , M. B. et
`al . , Lab Chip 16 ( 14 ) : 2719-29 ( 2016 ) ; Loskill , P. et al . , Lab
`Chip 17 ( 9 ) : 1645-54 ( 2017 ) ; Phan , D. T. T. et al . , Lab Chip
`17 ( 3 ) : 511-20 ( 2017 ) , each of which is incorporated herein
`by reference in its entirety ) . However , due to difficulties in
`maintaining the mature phenotype of adult human cardiac
`tissue in vitro , thus far there has not been a heart - on - a - chip
`system that supports long term organotypic culture of the
`human cardiac tissue .
`[ 0011 ] Thus , there remains an unmet need for systems and
`methods for achieving long - term culture of tissue slices
`while maintaining a specific phenotype of the tissue slice , in
`particular for maintaining an adult cardiomyocyte pheno
`type in human cardiac slices . Disclosed herein are culture
`systems and methods of using the same for organotypic
`culture of tissue slices . In some preferred embodiments , the
`culture system is a human - heart - on - a - chip culture for orga
`notypic culture of human cardiac tissue slices . The pro
`longed culture of tissue slices ( e.g. , human cardiac slices )
`demonstrated here allows for the study of chronic drug
`effects , gene therapies , and gene editing . To achieve long
`term culture and to minimize tissue differentiation , the
`culture system is an automated system which supports media
`circulation , oxygenation , temperature control , electrical
`stimulation , and mechanical loading . The culture parameters
`can be individually adjusted to establish the optimal culture
`condition . The culture system is also entirely self - contained
`to allow for the transport of live cardiac slices to share for
`scientific research and drug testing .
`
`BRIEF SUMMARY OF THE INVENTION
`[ 0012 ] Human cardiac slices have emerged as a promising
`model of the human heart for scientific research and drug
`testing . Retaining the normal tissue architecture , multiple
`cell type environment , and the native extracellular matrix ,
`human cardiac slices faithfully replicate the organ - level
`adult cardiac physiology without dedifferentiation .
`
`[ 0013 ] As described herein , organotypic culture condi
`tions have been optimized to maintain normal electrophysi
`ology of the human cardiac slices long - term , and an auto
`mated , self - contained heart - on - a - chip system has been
`developed for maintaining tissue viability and for transport
`ing live tissue . The prolonged culture of human cardiac
`slices described herein allows for the study of chronic drug
`effects , gene therapies , and gene editing . To achieve long
`term culture and to minimize tissue dedifferentiation , the
`culture system described herein supports media circulation ,
`oxygenation , temperature control , electrical stimulation , and
`mechanical loading . The culture parameters can be individu
`ally adjusted to establish the optimal culture condition . The
`heart - on - a - chip technology described herein further facili
`tates the use of organotypic human cardiac slices as a
`platform for pre - clinical drug testing and research in human
`cardiac physiology .
`[ 0014 ] The present disclosure provides a system for main
`taining and analyzing tissue slices , wherein the system
`comprises : a microfluidic device comprising at least one
`tissue culture chamber ; one or more actuators disposed in an
`interior of the tissue culture chamber ; one or more sensors
`disposed in the interior of the tissue culture chamber ; and an
`electronics module comprising a microcontroller , wherein
`the electronics module is coupled to the one or more
`actuators and the one or more sensors by an array of
`electrodes .
`[ 0015 ]
`The present disclosure further provides a method
`for maintaining one or more tissue slices in a microfluidic
`device , the method comprising culturing the one or more
`tissue slices in the microfluidic device , wherein the tissue
`slices are obtained from one or more organs , and wherein the
`microfluidic device comprises : a ) one or more tissue culture
`chambers , wherein each tissue culture chamber provides a
`restricted environment supplied with oxygen and nutrients
`necessary to maintain a desired phenotype for each tissue
`slice ; b ) one or more actuators for maintenance of the one or
`more tissue slices or for phenotypic interrogation of the one
`or more tissue slices ; c ) one or more sensors for measuring
`one or more physiological parameters of the one or more
`tissue slices ; and d ) an electronics module comprising a
`microcontroller , wherein the electronics module is coupled
`to the one or more actuators and the one or more sensors by
`an array of electrodes .
`[ 0016 ] Further objects and advantages of the present
`invention will be clear from the description that follows .
`BRIEF DESCRIPTION OF THE DRAWINGS
`[ 0017 ] Figures ( FIGS . 1 and 2 show that human cardiac
`slices remained viable for six days in culture , which allows
`for long - term pharmaceutical testing and evaluation of gene
`therapies . Human cardiac slices were cultured with 3 mL of
`medium in 6 well dishes placed on an orbital shaker at 20
`rpm in a tri - gas incubator with 5 % CO2 and 30 % 02. The
`culture medium was comprised of Medium 199 ( M4530 ,
`Sigma - Aldrich , St. Louis , Mo. ) , lx Insulin - Transferrin - Se
`lenium ( ITS ; 13146 , Sigma - Aldrich , St. Louis , Mo. ) liquid
`media supplement , 2 %
`penicillin - streptomycin ( P4333 ,
`Sigma - Aldrich , St. Louis , Mo. ) . Culture medium was
`replaced every two days . Activation maps were obtained
`from optical mapping experiments for conduction velocity
`calculations at day 0 ( FIG . 1A ) , day 2 ( FIG . 1B ) , day 3 ( FIG .
`1C ) , and day 4 ( FIG . 1D ) in culture . FIG . 2 shows conduc
`tion velocity of culture human cardiac slices at 1,000 mil
`
`26
`
`

`

`US 2019/0376014 A1
`
`3
`
`Dec. 12 , 2019
`
`liseconds ( ms ) pacing cycle length . The data are shown as
`mean - standard deviation . The statistical analysis was per
`formed using one - way ANOVA followed by a Dunnett's
`multiple comparisons test .
`[ 0018 ] FIG . 3 depicts a block diagram of a microfluidic
`system for organotypic culture of cardiac slices . This
`dynamic culture system can be used to maintain tissue slice
`viability and to prevent tissue dedifferentiation .
`[ 0019 ] FIG . 4 depicts a top view ( FIG . 4A ) and a side view
`( FIG . 4B ) of a tissue culture chamber which provides a
`restricted environment supplied with oxygen and nutrients
`necessary to maintain a desired phenotype for a tissue slice
`long - term .
`[ 0020 ] FIG . 5 depicts the configuration of two or more
`tissue culture chambers ( e.g. , microfluidic chambers ) in
`series ( FIG . 5A ) , parallel ( FIG . 5B ) , or combinations thereof
`( FIGS . 5C - D ) .
`[ 0021 ] FIGS . 6 and 7 are block diagrams depicting the
`interface between a culture system and a control unit ( e.g. ,
`a microcontroller ) . One or more tissue culture chambers of
`the culture system are instrumented with an array of actua
`tors and sensors which allows the control unit to monitor and
`control components of the culture system via a wireless or
`wired communication . For example , the control unit can
`actuate the delivery of a pharmacological compound or
`therapeutic virus contained within the media of a media
`reservoir to a tissue culture chamber when specific physi
`ological parameters are met within the tissue culture cham
`ber .
`[ 0022 ] FIGS . 8 and 9 show that oxygenation and circula
`tion of culture medium improve tissue viability . FIG . 8
`shows the recovery of murine sinus rhythm with medium
`circulation . FIG . 9 shows the recovery of murine sinus
`rhythm with medium oxygenation .
`[ 0023 ] FIG . 10 depicts a microfluidic culture chamber
`designed for in vitro cell type reprogramming and functional
`characterization of cardiac slices .
`[ 0024 ] FIGS . 11-13 show that human cardiac slices
`remained viable for 21 days in culture . FIG . 11 shows the
`conduction velocity ( cm / s ) of cultured human cardiac slices
`over time . No significant changes in conduction velocity are
`observed in human cardiac slices cultured for two days and
`four days when compared with fresh human cardiac slices .
`The data are shown as mean standard deviation . The sta
`tistical analysis was performed using one - way ANOVA
`followed by a Dunnett's multiple comparisons test . FIG . 12
`shows a comparison of action potentials of a fresh human
`cardiac slice ( “ Day 0 ' ) and a human cardiac slice cultured
`for 21 days ( “ Day 21 " ) . FIG . 13A shows an activation map
`of the fresh human cardiac slice . FIG . 13B shows an
`activation map of the human cardiac slice cultured for 21
`days ( “ Day 21 ” ) . The bars adjacent to the activation maps of
`FIGS . 13A and 13B represent activation times in millisec
`onds ( ms ) . FIG . 13C shows an action potential duration
`( APD ) map of the human cardiac slice shown in FIG . 13A .
`FIG . 13D shows an APD map of the human cardiac slice
`shown in FIG . 13B .
`[ 0025 ] FIG . 14 depicts a block diagram of a self - contained
`culture system . The culture system maintains tissue slice
`viability by providing optimal medium circulation , oxygen
`ation , and temperature control . To prevent tissue remodeling
`and dedifferentiation , the culture chambers are instrumented
`with pacing electrodes for electrical stimulation . The culture
`system is controlled by a microcontroller ( e.g. , Teensy 3.2 ) .
`
`Power is supplied by a battery ( e.g. , a 5V 30,000 mAh
`battery ) . Medium circulation is driven by a pump ( e.g. , a
`low - power , multichannel syringe pump ) . Medium tempera
`ture and oxygenation is maintained by a custom gas
`exchanger and heater unit . Temperature sensors are built into
`the culture chambers for feedback control . A multichannel
`ECG can be measured simultaneously .
`[ 0026 ] FIG . 15 illustrates an example of an assembled
`culture system . Insulation foam was removed from the sides
`of the enclosure for illustration of the assembled culture
`system .
`[ 0027 ] FIG . 16 illustrates example components of a cul
`ture system . FIG . 16A shows an example of a modular
`electronic control unit . The electronic components of the
`modular electronic control unit comprise , from left to right ,
`an acquisition module ( for ECG and temperature sensing ) , a
`motor driver ( e.g. , a pump driver ) , a microcontroller , and a
`power management module ( for supplying suitable voltages
`required for electrical stimulation and medium temperature
`control ) . FIG . 16B illustrates a fully integrated device that is
`designed for compactness and comprises a microcontroller ,
`an ECG module , a solution inlet , a solution outlet a ther
`moelectric heater / cooler , and three tissue culture chambers
`disposed in series . FIG . 16C illustrates a tissue culture
`chamber instrumented with sensing electrodes , field pacing
`electrodes , a temperature sensor , and an LED .
`[ 0028 ] FIG . 17 illustrates an example of a culture system
`that is modular in design for ease of scaling up culture
`capacity .
`[ 0029 ] FIG . 18 shows a recoded waveform during elec
`trical field stimulation with 3V pulse amplitude , 5 ms pulse
`duration , and 1 second pacing cycle length .
`[ 0030 ] FIGS . 19-23 provide an overview of the custom
`gas exchanger , medium heater , and pump ( for maintaining
`culture medium oxygenation , temperature , and circulation )
`of the culture system . FIG . 19 illustrates a CNC milled gas
`exchanger . The top chamber is made of polycarbonate for
`liquid medium to pass through . The bottom chamber is made
`of stainless steel and is heated with a thermofoil heater . FIG .
`20 depicts a 3D printed peristaltic pump . FIG . 21 depicts a
`portable gas tank and miniature pressure regulator . FIG . 22
`shows recorded temperatures of the heater and the culture
`chamber . The culture chamber temperature rapidly reached
`and maintained physiological temperature with minimal
`fluctuation . FIG . 23 shows the dissolved oxygen level in
`culture medium with different gas . The oxygen concentra
`tion in the liquid medium rapidly reached saturation when
`the gas exchanger was filled with oxygen . The dissolved
`oxygen was depleted when the gas exchanger was filled with
`nitrogen .
`[ 0031 ] FIGS . 24-27 relate to organotypic culture of human
`and murine cardiac tissue in a heart - on - a - chip culture sys
`tem . FIGS . 24A - C show activation maps of an acute human
`cardiac slice and slices cultured for 1 and 3 days in the
`culture system . The bars adjacent to the activation maps of
`FIGS . 24A - C represent activation times in milliseconds
`( ms ) . FIG . 25 shows the action potential recorded from the
`slices using optical mapping . The heart - on - a - chip system
`maintained a stable heart rate of the cultured murine atrial
`preparation ( FIG . 26 ) . FIG . 27 shows the far - field recording
`of the cultured murine atrial preparation .
`[ 0032 ] FIG . 28 depicts custom monitoring and analysis
`software . The main graphical user interface has 4 sections .
`Section 1 is for loading the device log and for selecting ECG
`
`27
`
`

`

`US 2019/0376014 A1
`
`4
`
`Dec. 12 , 2019
`
`recordings . Section 2 shows the history of the device