Laminar Wash™ AUTO system: a reliable walk-away sample preparation solution for better TIL recovery without centrifugationKim I1, Lye M1, Eberle C2, Ke CY1, Fernandez Oropeza N1, Wang A1, Cheng KL1, Kon IC 1, Pek R1, Kim N1

Curiox Biosystems Inc., MA, USA1, Charles River Laboratories, MA, USA2

AUTOMATED LAMINAR WASH™ PRODUCES CONSISTENT DATA with LOW COEFFICIENT of VARIANCE

Fig 2 By using Laminar Wash™ in a single wash step during immunophenotyping of human PBMCs, the user achieved (a) consistency and (b) lower coefficient of variance even between different operators. Webinar available from www.curiox.com

Conventional method ~20% CV

Population % CV

Monocytes 1.5

TCRαβ 1.0

B cells 3.4

NK cells 1.2

Vδ1 11.1

Vδ1-Vδ2- 10.1

Vδ2 2.4

LAMINAR WASH™ ENABLES ACCURATE IDENTIFICATION of TUMOR INFILTRATING LYMPHOCYTE (TIL) POPULATION

Fig 6(a) Centrifuge wash method – cell loss and mechanical stress through sequential pelleting and resuspension Fig 6(b) Laminar Wash™ method – less tumor debris, but higher retention of TILs and better resolution of populations

Single cells Live cells CD45+ cells CD3+ cells

ACKNOWLEDGEMENTSWe would like to give special thanks to the following scientists: Dr. Arnaud Colantonio at AdicetBio (Fig 2), Dr. Christoph Eberle at Charles River Laboratories (Figs 4&6), Blanca Ponce-Ngo at Montefiore Medical Center in NY (Fig 5), Dr. Thomas Tan, Jonathan Hsu, and Benjamin Keller Elstar Therapeutics (Fig 7), Dr. Jörgen Adolfsson at Lingköping University (Fig 8) for sharing their valuable insights and data. For webinars, please visit www.curiox.com.

LAMINAR WASH™ LEADS to IMPROVED CELL VIABILITY of TILs and LESS CYTOMETER CLOGGING

CURIOX’S LAMINAR WASH TECHNOLOGY AND FULL AUTOMATION OF SAMPLE PREPARATION WITH LW™ AUTO1000

(a) (b)

Fig 1 Curiox’s centrifuge-less cell preparation platform is enabled by our wall-less plate and laminar flow washer.

(a) The Laminar Wash™ (LW) plate (96 well) or strip (16 well) consists of an array of hydrophilic spots surrounded by hydrophobic surface, which functions as a virtual wall that separates each spot. Each spot can process from a single cell to as many as 10 million cells without the mechanical stress and cell losses associated with centrifugation.

(b) HT1000 washes 96 wells in a fully automated process with individual nozzles for each well.

(c) The Laminar Wash™ AUTO 1000 software interface allows modifications to sample volume, buffer volume, antibody volume, number of washes, incubation time and temperature. In addition, it comes with pre-programmed protocols to ensure user consistency. User-friendly Graphical Interface (GUI): The software prompts the user to conveniently enter the parameters of existing SOPs or protocols with no coding or programming of the liquid handling system.

0%#

1%#

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Monocytes#

TCRαβ#

B#cells#

NK#cells#

Vδ19Vδ29#

Vδ1#

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Operator#1#

Operator#2#

Operator#2#

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Operator#1#

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Perc

ent o

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TCRαβ#

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Operator#1#

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Operator#2#

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(a) (b)

Fig 7 FACS plots by (a) centrifuge vs. (b) Laminar Wash™ methods. The dissociated tumor samples from Raji-inoculated Balb/c mice showed higher viability and increased debris removal with Laminar Wash™, leading to less clogging and more consistent reading.

Cent

rifug

eLa

min

ar W

ash™

Cent

rifug

eLa

min

ar W

ash™

(a)

(b)

(a)

(b)

All events Live Single cells hCD45+

Live50.0

Debris9.04

Dead 28.0

Live73.6

Debris7.17

Dead 14.8

Live

ꟷAcquisition time ꟷ>

ꟷAcquisition time ꟷ>

SSC-A

FSC-H

SSC-A

mCD45

17.7%

28.9%

48.0%

hCD879.5%

70.4%

23.3%

SSC-A

SSC-A

FSC-H

SSC-A

mCD45

52.2%

34.5%

51.3%hCD8SSC-A

80.7%

20.7%

71.2%

SSC-A

FSC-H

SSC-A

mCD45

36.8% 19.0%

53.2%hCD8SSC-A 93.8% 17.1%

60.0%

FSC-A

SSC-A

FSC-A

FSC-H

L/D

SSC-A

hCD45

mCD45

47.4% 3.68%

76.6%

hCD4

hCD8

hCD3

SSC-A95.5%

11.8%

85.4%

Live47.4%

Live36.8%

Live52.2%

Live17.7%

LAMINAR WASH™ INCREASES % LIVE CELLs for TILs and LEADS TO HIGHER hCD45+ POPULATION

hCD4548.0%

hCD4551.3%

hCD4553.2%

hCD4576.6%

Cent

rifug

eLa

min

ar W

ash™

Cent

rifug

eLa

min

ar W

ash™

Live hCD45+ CD3+

Fig 4 A549-inoculated humanized mouse (a) unchallenged tumors (b) challenged tumors

(a)

(b)

(c)

LAMINAR WASH™ IMPROVES RETENTION and BETTER RETAINS ENDOGENOUS PROTEIN EXPRESSION

(a) (b)

Centrifuge Laminar Wash™

Fig 3(a) In processing of low cell numbers (50-100), LW retained higher cell count with better cellular morphology and brighter endogenous fluorescent expression. The dim protein expression is evidence of mechanic stresses caused by centrifugation.Data from a biotech company in San Diego

Fig 3(b) In subcutaneous murine melanoma tumor samples, LW retained 40% more live cells than centrifuge, with less tumor debris and better antibody staining.

tota

l live

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live C

D45+

0.0

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l rec

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Laminar WashTM

Centrifuge

** **

Cells settle on the Plate and washed in BarCode perm 35 min

Cells incubated with Barcode and washed in CSB 35 min

Cells incubated with blocking and surfaceantibodies washed in CSB 40 min

Cells fixated in 4% paraformaldehyde and incubated overnight

Cells incubated with Iridium and washed in CSB 35 min

Cells washed in ddH2O 35 min

Total time: 180 min

Total time: 225 min

Samples pooled and injected into the CyTOF

Cells resuspended in Barcode perm and washed twice in CSB

Centrifugation20 min

Cells incubated with Barcode and washed twice 50 min

Cells incubated with blocking and surfaceantibodies washed twice in CSB

55 min

Cells fixated in 4% paraformaldehyde and incubated overnight

Cells incubated with Iridium and washed twice in CSB

50 min

Cells washed twice in ddH2O 50 min

LAMINAR WASH™ BRINGS TIME SAVINGS TO THE OVERALL WORKFLOWLaminar Wash

Fig 8 Overall processing time for the CyTOF samples by (a) centrifuge vs. (b) Laminar Wash™

(a) (b)

LAMINAR WASH™ DECREASED BACKGROUND in B CELL CROSSMATCH without PRONASE TREATMENT

Fig 5 Inter-operator results versus Laminar wash – Analysis of tumor-bearing tissue from mice treated produced varying degrees of debris which Laminar wash efficiently reduces. Manual 1 and 2 centrifuge-based washing represents different experiences of operator-based processing. Live cell staining indicates better resolution achieved with non-pelleting wash of tumor cells.

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Tumor-naive_debris

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Laminar WashTM

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Viability

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%debris

Tumor-naive_debris

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Laminar WashTM

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Viability

Tumor-naive

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1×106 2×106 5×1060

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%debris

Tumor-naive_debris

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Manual 2

Laminar WashTM

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Viability

Tumor-naive

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Total events, 2×106 cells per sample

Single cells, 2×106 cells per sample

Manual 1 Manual 2 Laminar Wash™