Three-dimensional (3D) tumor cell culture models have been shown to represent a biologically relevant assay system. We've optimized methods to culture Patient derived Primary tumor cells in 2D & 3D formats. Over 20 + Gliomas and 40+ Breast cancer samples from unique patients have been cultured. These cultures are also enriched in Cancer stem cells ! Pls contact for more details: Rachna@saarum.com +91 7032647554

1. Bio-Bank & Personalized Medicine
Primary cell models – phenotypic assays
Research & Clinical Support Services
A joint venture with
23-Oct-15 1

2. The ‘Essence’ of our Enterprise
23 October 2015 2
Vision: To be a premier global biotech that employs human translational platforms for the
discovery & development of novel diagnostics, biomarkers & drugs with better clinical
outcomes
Mission:
• Build a systematic archive of ethically consented,
anonymized human samples & associated data for
developing novel biomarkers & diagnostics
• Leverage primary human tissues & stem cells for disease
modeling, target & drug discovery with higher rates of
clinical success
• Use of tissues for R&D, NOT for regenerative purpose
Saarum Sciences & Sapien Biosciences were initially set-up as two separate entities. We are in the process of merging both operations into Sapien Biosciences for better synergy

3. 23-Oct-15 Introduction to Saarum-Sapien 3
Data Associated
With Our Samples
Current Inventory: >0.1 M Samples; Projected Inventory (in ~3 years): ~0.5-1M Samples
Sample diversity & associated data at Sapien

4. 23-Oct-15 Introduction to Saarum-Sapien 4
Sample formats available
Viable/Live Cell Types Available
Sample Formats Available
Fresh: Live/snap-frozen

5. R&D Infrastructure available to Sapien
23-Oct-15 Introduction to Sapien Biosciences 5

6. PBMCs, B/T cells,
CD34+, HUVECs,
fibroblasts etc.
In vitro cell-based
assay systems, models
6
Breast Cancer,
Glioma, Head & Neck
cancers etc.
ID & validation of
drug targets &
Companion Dx
Breast Cancer,
Glioma, Prostate
Cancer etc.
Lead-optimization of
novel cancer-active
molecules
Breast cancer model
with in-built readouts
Screening drugs for
metastasis & cancer
stem cells
Cytokine release,
Chemoattraction,
Migration etc.
Phenotypic screening
of novel drugs
Current
Offerings
Applications
Patient samples and
associated data
OncoBloc™ OncoPrime™ MetBlock™ TruCell™ TruScreen™
Fixed & frozen
samplecollections
Patient-derived live
cancer cells
Novel model of
metastasis (EMT)
Patient/Normal
cells, derivatives
Disease-relevant
functional assays
Platform
Our translational research products and platforms

7. 23-Oct-15
OurGlioma
collection
Calcein (0.5 uM) PI (0.5 uM) Merged
10 X 10 X 10 X
10 X 10 X 10 X

8. 23-Oct-15
Glioma Patients Samples and data
107 4242
Total no. of Glioma cases n = 149
FFPE cases
Fresh frozen
Grade* No.
I 2
II 19
III 7
IV 13
Gender
Male 22
Female 20
Year wise
distribution of 42
cases
No of cases in
each year
Availability of
FFPE blocks for
the no of cases
2013 18 17
2014 23 21
2015 1 1

9. 23-Oct-15
Available data set with the cases
Patient Demographics
1 Gender
2 Race
3 Nationality
4 Age
Surgery details
1 Date of Surgery
2 Surgical Procedure
3 Surgical Margin (if any)
4 Location
Chemotherapy details
1 Date of Chemotherapy
2 Date of Chemotherapy Ended
3 Drug Name
4 Number of Cycle
5 Location
Radiotherapy details
1 Date of Radiotherapy
2 Date of Radiotherapy Ended
3 Radiation volume, fraction, site
Outcome details
1 Date of Last Contact
2 Vital Status
3 Cancer Status
4 Cause of death
5 Death Status
6 Recurrence, if any and type

10. 23-Oct-15
Age distribution, Recurrence, samples in culture
1
3
15
7
8
5
3
No of Patients n = 42
0 to 20 yrs
20 to 30 yrs
30 to 40 yrs
40 to 50 yrs
50 to 60 yrs
60 to 70 yrs
70 and Above yrs
Tissue samples =
42
Data available =
37
Newly
diagnosed
cases = 30
Recurred
cases = 7
Recurred
samples with old
blocks = 2

11. 23-Oct-15
Treatment Information for the 7 Recurred Cases
at onset and during recurrence respectively
Treatment No of Patients
Only Surgery 1
Surgery + Adj. CT + RT 3
Surgery + Con. CT + RT 3
1
3
3
No of Patients
Only Surgery
Surgery + Adj. CT
+ RT
Surgery + Con.
CT + RT
Treatment No of Patients
Only Surgery 1
Surgery + RT 1
Surgery + Con. CT + RT 5
1
1
5
No of Patients
Only Surgery
Surgery + RT
Surgery + Con. CT +
RT

12. 23-Oct-15
Recurrent samples – details
SB IDs Age Sex Diagnosis
Type of
Glioma
Site of
tumour
Type of Surgery
Chemotherapy:
Drugs/Regimen/
No of Cycles
Concurrent
Chemotherapy
Radiation
Therapy:
Type/Dose/Fracti
ons
Is Serum
Available?
SB00008528 54 F
Rt Fronto
temporal
glioma
Astrocytoma
Grade II
Rt Fronto
temporal
Rt Frontotemporal
Craniotomy and
excision of tumour
CT not given
Cap
Temozolomide -
100 mg
Ext : 5580 cGY/31
frs
No
SB00006077 35 F
Lt Temporal
glioma
Anaplastic
Astrocytoma
Grade II
Lt Temporal
region of
brain
Lt Temporal
Craniotomy in Lt
insular region
CT not given
Concurrent CT
not given
Ext : 3600 cGy/20
frs
No
SB00005972 45 F
Rt Insular
glioma
Anaplastic
Astrocytoma
Grade II
Rt Insular
region of
brain
Rt Insular
Craniotomy and
Parietal excision of
tumour
Adj : Cap
Temozolomide x 5
days for cycles
Concurrent CT
not given
Ext : 5580 cGY/31
frs
No
SB IDs Type of Tumour for Recurrence
Site of
Recurrence
Type of Surgery at
Recurrence
Concurrent Chemotherapy :
Drugs/ Regimen (Treatment
at Recurrence)
Radiation Therapy:
Type/Dose/Fractions(Tr
eatment at Recurrence)
Is Serum
Available?
SB00008528 Glioblastoma Multiforme, Gr - IV
Rt Fronto
temporal
Rt Frontotemporal
Craniotomy and
excision of frontal
lesion
Cap Temozolomide - 120 mg
during RT
Ext : 5800 cGY/ 329 frs No
SB00006077 Anaplastic Astrocytoma Gr - III
Lt Fronto
temporal
region of
brain
Lt Fronto-temporal
Craniotomy and Ant
Temporal lobectomy
Concurrent CT not given Ext : 5040 cGY/ 28 frs No
SB00005972 Anaplastic Astrocytoma Gr - III
Rt Insular
region of
brain
Redo Craniotomy
and gross toal
excision of tumour
Cap Temozolomide - 120 mg
during RT
Ext : Dose PH- I- 4500 cGY/
25 frs and PH-II - 5580
cGy/31 frs
No
OriginalTumorRecurrentTumor

13. 23-Oct-15
Glioma primary cultures - ~ 20 patient samples cultured
No Sapien ID Pathology Age (Sex) Serum Plasma DNA FFPE
#1 SB.0000305 Gemistocytic astrocytoma Grade II 58 (M) Yes No No Yes
#2 SB.00000474 Anaplastic Oligodendroglioma WHO Grade III 49 (M) No Yes No Yes
#3 SB.0006129 Anaplastic Astrocytoma WHO Grade III 32 (M) No No Yes Yes
#4 SB.0000295 Anaplastic Astrocytoma, WHO Grade III 36 (F) Yes No No Yes
#5 SB.0006077 Anaplastic Astrocytoma WHO Grade III 35 (F) No No Yes (*RT) Yes (*RT)
#6 SB.0005972 Anaplastic Astrocytoma, WHO Grade III 45 (F) No No No
6 *OT + 2
*RT
#7 SB.0008528 Glioblastoma Multiforme, WHO Grade IV 54 (F) No No No
Yes
(*RT)
#8 SB.0005980 Glioblastoma Multiforme, WHO Grade IV 56 (M) No Yes No Yes
#9 SB.0000298 Glioblastoma Multiforme, WHO Grade IV 35 (F) No No No Yes
#10 SB. 0000038 Glioblastoma Multiforme WHO Grade IV 58 (M) No No No Yes
#11 SB.0000085 Glioblastoma Multiforme, WHO Grade IV 54 (F) Yes Yes Yes Yes
#12 SB.0000144 High Grade Glioma 38 (F) No Yes Yes No
Note : Glioma samples marked with YELLOW are recurrent patient samples.
*OT – Original Tumor, *RT – Recurrent Tumor

14. Cell lines are poor model of cancer leading to drug failures
CD133 +ve cells : 35%
CD133 +ve cells : 1.5 %
CD133 +ve cells : 11%
CD133 +ve cells : 2%
Monolayer
culture (2D)
Neurospheres
(3D)
U87MG
SB.30648
Patient derived
culture
Observation: Flow cytometry detection of cancer stem cells (CSCs) by CD133-PE antibody staining in U87MG cells and glioma patient-derived cells in
both monolayer and neurosphere cultures. The percent CD 133 positive cells for 2D cultures were 2% (U87MG) and 11% (SB.30648). Whereas percent
CD133 positive cells for 3D cultures were 1.5% (U87MG) and 35% (SB.30648).
The low percent of CD133 positive cells for U87MG in 3D cultures were also showed by Vacas-Oleas et al (vol 2 , issue 1, 2013) published in Open
Access Scientific reports.
U87MG
SB.30648
Patient derived
culture

15. 23-Oct-15
Glioma cultures in 2D - GBM (SB.8528) & U87MG Cell Line
SB.8528
100 µm
2D SB.8528
100 µm
2D
Day 4 Day 6
U87MG
200 µm
2D
Day 10
U87MG
200 µm
2D
Day 15
Rate of Proliferation
SB.8528 28 h
U87MG 24 h

16. 23-Oct-15
Characterization of Glioma cultures: Localization of Beta-III Tubulin
Culture: High Grade Glioma (SB-144)
Antibody Staining: Beta-Tubulin
A B
Observations: Beta-III-tubulin is major
constituent of microtubules. In the
above figure beta-III-tubulin
immunofluorescence staining is
observed on cytoskeleton of most cells.
Primary Ab: Mouse
Anti-Tubulin, beta III
(Millipore)
Ab Concentration;
250ug/ml
Ab dilution used: 1:100
Secondary Ab: Goat
Anti-Mouse IgG
AlexaFluor 488(Green)
(Molecular Probes)
Ab Dilution: 1:1000
DA
PI
20 X
No 1°
Ab
Introduction to Sapien and Saarum

17. 23-Oct-15
Localization of Glial Fibrillary Acidic Protein by Immunofluorescence
Culture: Anaplastic Astrocytoma (SB.6129)
Antibody Staining: GFAP
A B
Primary Ab: Purifed
Mouse Anti-GFAP
(BD Inc)
Ab dilution 1:100
Secondary Ab: Goat
Anti-Mouse IgG
AlexaFluor 488
(Green)
(Molecular Probes)
Ab Dilution; 1:1000
DA
PI
No 1°
Ab
Observations: GFAP is
an intermediate
filament protein present
in astrocytes. GFAP is
co-localized in
cytoskeleton of the cells.

18. Glioma cultures - 3D - High grade Glioma (SB.144) & U87MG
SB.144 SB.144
10X 10X
10X 10X
3D U87MGU87MG 3D
3D 3D
We have successfully cultured
high grade gliomas (SB.144
shown here) and U87MG cells in
serum free media on non-treated
plates for 10 to 15 days to form 3
Dimensional spheres known as
neurospheres. Such neurospheres
better represent human tumor
biology in vitro and can be used
for drug screening.
CSC evaluation is underway

19. Fluorescent staining of Anaplastic astrocytoma neuro-spheres
(SB.6129) by Calcein-AM & PI
Calcein (0.5 uM) PI (0.5 uM) Merged
10 X 10 X 10 X
Calcein (1 uM)
10 X
PI (0.5 uM)
10 X
Merged
10 X
Observation: Anaplastic astrocytoma (SB.6129) cells were cultured in non-treated 6 cm dishes in serum-
free medium as spheroid cultures (neurospheres) for 7 days. Then, neurospheres from Passage 2 were
stained with Calcein-AM and Propidium Iodide to detect the live (Green, Calcein-AM) and dead (Red, PI)
cells within neurospheres. Image shows that more than 95% of cells in neurospheres were alive.

20. H & E staining of SB.6129 neurospheres as Paraffin embedded
spheroids
Observation: Anaplastic astrocytoma cells (SB.6129) were cultured in non-treated 6 cm dishes in serum-
free medium as spheroid cultures (neurospheres) for 7 days. Then, egg albumin cell blocks were made
with these neurospheres, followed by formalin fixation and processing for paraffin block preparation.
Image above is an H&E stained section from the block showing a group of tumor cells with well preserved
nuclei and cytoplasm. Dead cells stain black. Only 4-5 cells appear to be dead in the entire neurosphere
confirming >95% cells being viable, as also shown by calcein staining in the previous slide.
Arrow indicates
dead cells stained
black

21. Comparison of CD 133 +ve cells (cancer stem cells) between monolayer (2D) vs Neurospheres (3D)
cultures in GBM cell line (U87MG) & Desmoplastic infantile Ganglioglioma, Grade-I (SB.30648)
CD133 +ve cells : 35%
CD133 +ve cells : 1.5 %
CD133 +ve cells : 11%
CD133 +ve cells : 0.3%
CD133 +ve cells : 2%
CD133 +ve cells : 0.6 %
Monolayer culture
(2D)
Neurospheres
(3D)
U87MG
U87MG
SB.30648SB.30648
40 X
U87MG
U87MG
2D
100 X
3D
100 X
SB.306482D
100 X
SB.306483D
Observation: Flow cytometry detection of cancer stem cells (CSCs) by CD133-PE antibody staining in U87MG cells and glioma patient-derived cells in both monolayer and
neurosphere cultures. The percent CD 133 positive cells for 2D cultures were 2% (U87MG) and 11% (SB.30648). Whereas, percent CD133 positive cells for 3D cultures were 1.5%
(U87MG) and 35% (SB.30648). The low percent of CD133 positive cells for U87MG in 3D cultures were also showed by Vacas-Oleas et al (vol 2 , issue 1, 2013) published in Open
Access Scientific reports.
Unstained cells Unstained cells

22. Titrating a known Cytotoxic Drug in 3D Glioma cultures
Absorbance@450nm
Bendamustine conc (uM)
Dose Response Curve
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Observation: U87MG and one high grade glioma cell culture was
treated with Bendamustine at the concentrations indicated above. Dose
response curves were used to calculate IC50 values; the calculated IC50
value for both cell types was ~300 uM.

23. Dose-response of Compound B in Anaplastic Astrocytoma
Neurospheres
• Compound B concentrations tested were 468 nM, 938 nM, 1.8 uM,
3.75 uM, 7.5 uM, 15 uM & 30 uM.
• Drug treatment was performed for 3 days (72 h) in triplicate.
• After incubation, WST-1 was added to samples and incubated for 3 h.
• The absorbance was read at 420 nm & 630 nm.
Observation: Treatment of primary anaplastic astrocytoma neurospheres with Compound
B demonstrated significant inhibition of viability, with IC50 between 0.9-1.9uM.
0
0.1
0.2
0.3
0.4
Absorbance(420-630)nm
Drug Concentration (uM)
SB.6129 neurospheres treated with
Compound B
SB - 6129 Sample SD
Cells alone 0.36 0.01
468 nM 0.27 0.07
938 nM 0.18 0.05
1.88 uM 0.19 0.03
3.75 uM 0.12 0.04
7.5 uM 0.09 0.03
15 uM 0.1 0.06
30 uM 0.06 0

24. Migration assay for Anaplastic astrocytoma (SB.6129)
DMSO 0 h DMSO
40 X
DMSO
0 h Benda
(300 uM)
40 X
48 h24 h
24 h 48 h
Benda
(300 uM)
Benda
(300 uM)
40 X 40 X
40 X40 X

25. Migration assay for Anaplastic astrocytoma (SB.6129)
Observation: Treatment with Bendamustine (300 µM) effectively prevented the migration of anaplastic astrocytoma (SB.6129) cells at
24h & 48h. DMSO control showed filling of created open wound in 48 h.
98.33
47.65
93.18
5.94
96.88
DMSO DMSO BENDAMUSTINE DMSO BENDAMUSTINE
0 H 24 H 48 H
PercentageOpenWoundArea
Migration Assay1. Anaplastic astrocytoma (SB.6129) cells were plated in
6-well plate for wound healing assay (migration
assay).
2. Cells were grown to confluency in 6-well plate in their
regular growth medium. Cell cultures were scratched
with a 200 µl sterile pipette tip and washed with PBS
to remove detached cells.
3. Cells were treated with 300 µM Bendamustine
(duplicate) in parallel with 0.25% DMSO as control.
4. Images were acquired at 0h, 24h & 48h time points.
5. For automated image analysis, the acquired image
data set was analyzed with the TScratch software.

26. Other Biomarker & Drug discovery platforms & case studies
• FFPE’s preserve
TIL signature
and can be
studied at
molecular level
• Study specific TIL type(s) impacting
your compounds clinical outcome
60 %
• In house data showing positive
correlation between amount of TIL and
prognosis in TNBC & Her2 positive breast
cancer
Sample/Disease Diversity
• A diverse collection of
disease specific FFPE
• Suitable for discovery &
validation of novel
biomarkers & drug targets
OncoBloc™

27. Other Biomarker & Drug discovery platforms & case studies
• Physiological relevant
primary cells : available
frozen or in culture, ready
to be shipped & also custom
collection
TruCell™
Breast
Glioma
Prostate
Cervical
Leukemia
Lymphoma
Hematological
cancer
-Blood cells : PBMC, T/B
cells, Neutrophils
healthy & Patients
-Skin Cells- Keratinocytes,
Melanocytes, Fibroblasts,
-Tumor associated
fibroblast
-Synoviocytes
Cytotoxic effect of compound on B-cells
isolated from NHL patient
Neutrophil (from healthy donor)
activation assay
0
50
100
150
0.1 uM 1 uM 10 uM No Act
PercentMPOActivity
MPO assay for 0.1 million cells
per well
fMLP, 2h fMLP, 4h fMLP, 16 h
Cancer
PrimaryCells
Concentrationof
Cytarabine (µM)
Apoptoticcells
(%)
DMSO control 4.5
0.1 6.8
1 11.0
10 15.3

28. Other Biomarker & Drug discovery platforms & case studies
• Suitable to study
underlying biology
• For phenotypic screening
& optimization of novel
inhibitors of cancer
metastasis
MetBlock™
EPCAM & CK8/18 – Epithelial marker
Vimentin & SMA – mesenchymal markerEngineered Models
• Some early evidence on the
Epithelial to mesenchymal
transition in our patented system.
MetBlockTM

29. Other Biomarker & Drug discovery platforms & case studies
• Custom models using patient
cells to provide physiological
assay systems
• Suitable for screening
compounds for better clinical
outcome & to study action on
true disease phenotype.
TruScreen™ IL-17 inhibition in PBMC’s from COPD
patient
Skin organ ex-vivo culture
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
1nM 3nM 10nM 30nM 100nM 300nM 1uM 10uM
Percentageinhibition
Concentration ofX
Inihibition of IL-17expression by compound X
Patient 1
Patient 2
Patient 3
Day 0 Day 3
Day 5 Day 7
H&E stainingofskin tissueafter culturingfor specified number of days.
Suggests epidermis is intactand skin is maintained in good condition as also indicated by LDH

30. 23-Oct-15
Effe
OncoPrime™
3D culture High
grade Glioma
Neurospheres
• Cancer patient-derived primary
cell panel with true clinical
diversity & heterogeneity
• In-vitro phenotypic screening/
anti-proliferative activity of
compound on panel of diverse
cancer types
OurBreast
CancerPanel
Cell viability assay
with some S.O.C
Other Biomarker & Drug discovery platforms & case studies

31. 23-Oct-15
Sapien Biosciences Private Limited,
Saarum sciences Private Limited,
1st Floor, AIMSR Bldg. Apollo Health City,
Jubilee Hills, Hyderabad-500096
India
www.sapienbio.com
www.saarum.com
Phone: +91 7032647554
Contact us at: info@saarum.co.in
Phenotypic Screening Biomarker Discovery Custom Assays
Primary Cells FFPE
ANY QUESTIONS ?
Contact us