Investigating the immuno-modulatory effects of

hydrogels for immune cell culture

Donaldson, A1., Hall, L1., Vasanthi Bathri Narayanan, P1., Nikkhah, M2., Khademhosseini, A2., Ghaemmaghami, A1.
1Allergy
and Tissue Modelling Group, School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham, NG7 2UH, United
Kingdom. 2Brigham and Women's Hospital, Division of Biomedical Engineering, Harvard Medical School, Cambridge, MA 02139, USA
Introduction
There is a need for immuno-competent artificial tissue models to study key immunological processes in a physiologically relevant setting.
The development of such systems will improve scientific practise and contribute to reducing the use of animals in research.
The objective of this project is to develop a biologically relevant 3D culture system to mimic Illustration of the
interactions between dendritic cells and T cells which take place within lymph nodes in vivo. proposed lymph
node-like 3D
In addition to providing a platform for better understanding of DC-T cell cross-talk, the culture model to study
system could provide an alternative to the local lymph node assay used in chemical safety testing, immune
sensitization in
leading to a reduction of animal usage in this area
vitro.
Owing to their versatile physical and mechanical properties hydrogels are widely used as scaffolds in tissue engineering applications . Here we have used a
collagen based hydrogel to support the 3D cell culture.
Gelatin GelMA monomer Polymerised GelMA Gelatin methacrylate (GelMA) is a semi-natural hydrogel derived from collagen. Extensively
used for various tissue culture applications, routine toxicological readouts have
demonstrated its biocompatibility.

However, the immuno-compatibility of GelMA has not yet been characterised. Here, we
Chemical modification of gelatin by the addition of methacrylate present our findings from investigating the suitability of GelMA as a matrix for immune cell
groups to amine side chains gives rise to a photo-polymerizable culture.
hydrogel (J. Nichol 2010).
Methodology and Results
Human peripheral blood mononuclear cells (PBMCs) isolated from blood by density gradient
centrifugation over Histopaque layer. Cells seeded on photo-cured GelMA substrate (5%
3. GelMA suppresses the pro-inflammatory cytokine TNF-
GelMA, 0.25% photoinitiator) in complete RPMI-1640 media (10% FBS, 100 U/mL penicillin, Supernatants collected after 4 hours of culture with GelMA substrate -/+ LPS stimulation.
100mg/mL streptomycin, 2mM L-glutamine (all purchased from Sigma Aldrich). Cytokine analysis performed using the eBioscience flow cytometric multi-analyte detection system.
0.1g/mL E. coli lipopolysaccharide (LPS ) used for cell stimulation.
IL - 8 IL - 6
1. Viability is not compromised on a GelMA substrate 150
*
150 Figure 3. Immuno-
C y t o k in e p r o d u c t io n ( % )

C y t o k in e p r o d u c t io n ( % )
modulatory effects of
Figure 1. Cell viability on day 5 of culture
F lu o r e s c e n c e In t e n s ity ( 5 8 0 n m )

40000 100 100 GelMA on cytokine

with GelMA substrate is comparable
production. Detected
30000
with tissue culture plastic. 50 50
levels of LPS-induced
20000
TNF- are significantly
Cell health measured by alamarBlue. 0 0
10000
Error bars represent SEM. lower in cultures with
C

C
S

S
S

S
A

A
T

T
P

P
P

P
lM

lM

a GelMA substrate.
L

L
L

L
e

e
+

+
Two-tailed Students t test n=2 (ns= P > 0.05).
+

+
G

G
A

A
C

0
T

lM

lM
e

c
G

A e ti
lM u s
e s la
G is p IL - 1 T N F -
T
lt
u
re Data normalised to
u 150 150 ***
positive control (TC + LPS)
C y t o k in e p r o d u c t io n ( % )

C y t o k in e p r o d u c t io n ( % )

2. GelMA sequesters TNF- n=3 Error bars represent

100 ** 100
A range of concentrations of recombinant human TNF- were spiked into complete SEM 1 way ANOVA (* = P
RPMI media and added to GelMA substrate or a tissue culture plastic (TC) control. 0.05, ** = P 0.01, ***
After a 4 hour incubation at 37C to mimic tissue culture conditions, supernatants 50 50
= P 0.001, **** = P
were collected for cytokine analysis. 0.0001).
0 0
2000
D e t e c t e d T N F - ( p g /m l)

C
S

S
S

S
A

A
T

T
P

P
P

P
lM

lM
L

L
L

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+

+
+

+
G

TC
A

A
C

C
T

lM

lM

1500
e

G e lM A
G

Cells harvested after 30 minutes and 4 hours of culture with GelMA -/+ LPS stimulation to
1000
measure TNF- transcript levels by reverse transcriptase PCR.
Figure 2. A B
500
GelMA mops up soluble TNF-. 30 min 4 hr
Error bars represent SEM.
0 TNF-
0 .0 2 .5 5 .0

E x o g e n o u s T N F - ( n g /m L )
1 2 3 4 5 6 7 8
Summary GAPDH
Immune cell viability is not impaired by GelMA. 1 = TC 5 = TC
2 = TC + LPS 6 = TC + LPS
GelMA (but not gelatin; data not shown) seems to 3 = GelMA 7 = GelMA
sequester TNF- produced by PBMCs\ monocytes 4 = GelMA + LPS 8 = GelMA + LPS
in response to LPS stimulation.
Despite the initial up regulation of TNF- gene
expression seen at 30 minutes, the lack of soluble
TNF- in the microenvironment leads to
suppression of further gene transcription. GelMA binds TNF- thus inhibiting Figure 4. LPS-induced TNF- expression is attenuated at 4 hours of culture with a
a pro-inflammatory feedback loop. GelMA substrate. A. Real time quantitative PCR Ct Analysis (Normalised against
Hence, 4 hours after LPS stimulation there is low\ no TNF- protein detected in
GapDH). B. Ethidium bromide gel-based RT PCR. Representative of 2 data sets.
the supernatant, in addition to significant down regulation at gene level.
This is most likely due depriving cells from positive autocrine effects of TNF- on Acknowledgements References
TNF- gene transcription. National Centre for the 3Rs Nichol, J. W., Koshy, S. T., Bae, H., Hwang, C. M., Yamanlar, S., and
Miltenyi Biotec Khademhosseini, A. (2010) Biomaterials 31, 5536-5544.
Collectively this means that GelMA could have anti-inflammatory properties in an PhD supervisors: Felicity Rose, Cameron Alexander, Amir Ghaemmaghami Anderson, J. M., Rodriguez, A., and Chang, D. T. (2008) Seminars
Allergy & Tissue Modelling Group: P. Vasanthi Bathri Narayanan, F. Salazar., in immunology 20, 86-100
inflamed setting where there is usually high level of TNF- present. However, we Rushworth, S. A., Shah, S. and MacEwan D. J. (2011) J Immunol.
L. Hall.
conclude GelMA is not a suitable matrix for the proposed 3D DC-T cell culture Khademhosseini Lab: Mehdi Nikkah.
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