Evaluation of Dharmacon™ Accell™ siRNAdelivery into spheroids and hiPSCs
Allyson Malloy, Andrew Riching, Josien Levenga, Jan Korte, Anja van Brabant Smith, and Žaklina Strezoska
2650 Crescent Drive, suite 100, Lafayette, CO 80026

Introduction

AB
Relative Expression

Viability

1.2

1.2

11

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

00
Non-Targeting Control

Normalized Viability

Normalized Relative Expression

Accell siRNA Controls in hiPSCs

PPIB expression in HCT-116
spheroids grown in ULA plates

PPIB expression in LS174T
spheroids grown in ULA platesABHCT-116 Spheroids in ULAALS174T spheroids in ULA

B
1.2Expression, Fold vs NTC

Expression, Fold vs NTC

1.2
10.8
0.6
0.4
0.2
00% FBS

HCT-116 Medium

0% FBSAccell Medium

PPIB expression in HCT-116
D spheroids
embedded in GrowDex

PPIB expression in LS174T
C spheroids
embedded in Matrigel

0.8
0.6
0.4
0.2
01
0.8
0.6
0.4
0.2
0No Accell

PPIB

PPIB

96 hour

0.4
0.2

144 hourPPIB
96 hour

PPIB
144 hour

0% FBS
Accell Medium

1.2

10.8
0.6
0.4
0.20No Accell

0% FBSPPIB expression in LS174T
D spheroids
embedded in GrowDex

1.2Expression, fold vs. NTC

10.6

LS174T Medium

1.2Expression, fold vs. NTC

1.20.8

1% FBSExpression, Fold vs NTC

PPIB expression in HCT-116
C spheroids
embedded in Matrigel

101% FBS

10.8
0.6
0.4
0.2
0No Accell

PPIB
96 hour

PPIB
144 hourNo Accell

PPIB
96 hour

PPIB
144 hourFig 2. Fluorophore-labelled siRNA reveal siRNA uptake into HCT-116 spheroids,
resulting in strong knockdown of target mRNA. A) Uptake of fluorophore-labeled Accell
siRNA in HCT-116 spheroids formed in ULA plates. B) Low serum HCT-116 medium andAccell medium both result in knockdown of PPIB in ULA spheroids using Accell siRNA. C)
In spheroids formed in Matrigel, long term Accell siRNA treatment in Accell medium resulted
in PPIB knockdown. D) Spheroids grown in GrowDex also show strong knockdown after
treatment with Accell siRNA. NTC = Non-Targeting Control

Fig 3. Fluorophore-labelled siRNA reveal siRNA uptake into LS174T spheroids,
resulting in strong knockdown of target mRNA. A) Uptake of fluorophore-labeled Accell
siRNA in LS174T spheroids formed in ULA plates. B) Low serum LS174T medium andAccell medium both result in knockdown of PPIB in ULA spheroids using Accell siRNA. C) Inspheroids formed in Matrigel, long term Accell siRNA treatment in Accell medium resulted inPPIB knockdown. D) Spheroids grown in GrowDex also show strong knockdown after
treatment with Accell siRNA. NTC = Non-Targeting Control

Multiplexing Accell siRNA in HCT-116 spheroids
formed in ULA plates result in specific targetmRNA knockdown despite sequence homology

Experimental designs using Accell siRNA inthree different spheroid models

MAP2K1 Accell

AMAP2K2 Accell

MAP2K1 + MAP2K2 Accell

No Accell

0% FBSAccell medium

1.6
1.4
Expression, Fold vs NTC

Transfection-free RNAi with Accell siRNA intocomplex models

Successful target knockdown in LS174T
spheroids using Accell siRNAExpression, Fold vs NTC

Researchers are using more complex tissue models to study a variety ofdiseases, including hiPSC-differentiated cells and 3D spheroids. 3D tumorspheroid models offer numerous advantages over conventional twodimensional cell culture, which include the development of a heterogeneousarchitecture resulting in apical-basal polarity, and an internal gradient ofnutrient, oxygen, and signaling factors. Additionally, 3D models provide theability to study the biochemical and mechanical interaction of cells withextracellular matrix proteins. Therefore, tumor spheroids resemble tumors
grown in vivo more closely than 2D monolayer culture. Given the advantagesof 3D culture, we sought to determine if standard spheroid culture models
were susceptible to transfection-reagent free siRNA delivery using
Dharmacon Accell siRNA. We optimized culture conditions for threecommonly used spheroid models: seeding cells into ultralow attachment(ULA) plates, into Matrigel™, and into GrowDex®. We tested various serum
conditions, after recent literature reported that complexing siRNA in highserum can assist with siRNA delivery into spheroids. Furthermore, we tested
the efficiency of multiplexing Accell siRNA in HCT-116 spheroids by targeting
two genes simultaneously. Using these tools, we aim to guide researchers inthis field, who may be seeking guidance for siRNA delivery in complex cellculture models. In the future we plan to expand these studies to hiPSCderived organoids, such as brain and kidney. Developing robust protocols forsiRNA delivery in complex cell models will enable researchers to generate
reproducible results and will potentially advance the field of 3D cell culture.

Successful target knockdown in HCT-116
spheroids using Accell siRNAPPIB

1.2
1
0.8
0.6
0.4
0.2

Non-targeting control

PPIB

00% FBS

Accell medium
MAP2K1B

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Luminescence (RLU), Foldvs Untreated NTC

NTC

Fig 1. Transfection-free Accell siRNA reagent for complex models. A) Schematic of Accell
siRNA delivery using Accell delivery medium and added directly to cells without the need forlipid transfection or electroporation, allowing for RNAi in difficult-to-transfect cell types. Can beused with Accell delivery medium, as well as low serum medium. B) PPIB expression in WTChiPSCs was reduced by >90% compared to non-targeting control (NTC) without compromisingcell viability or iPSC morphology. Scale bar = 100 µm.

MAP2K2

MAP2K1

MAP2K2

MAP2K1 + MAP2K2

1.4
1.2
10.8
0.6
0.4
0.2
00% FBSAccellFig 4. Targeting the MAP2K1 and MAP2K2 genes simultaneously with Accell siRNA.
A) Successful mRNA knockdown 96h post transfection when multiplexing Accell siRNAtreatment targeting MAP2K1 and MAP2K2 genes with high specificity despite sequencehomology B) No effect on viability of cells 96h post transfection.

Conclusion
Here, we highlighted the successful use of Accell siRNA in threemodels of generating spheroids. Using Accell siRNA in combinationof GrowDex resulted in the strongest knockdown of the target genein spheroids generated either with the HCT-116 or the LS174 cellline. Multiplexing two Accell siRNA result in equal efficient
knockdown compared to a single Accell siRNA treatment. Combinedwith our extensive experience with synthetic RNA, these tools enable
the interrogation of genetic mechanisms in spheroids and hiPSC
using RNAi.