En Español

Services

PLACEMA is a technology platform offering standardized iPSC production services as well as subsequent differentiation into specific cell lines. Services cover requirements for both academic, clinical, and pharma applications.

PLACEMA was designed to offer high-end technology content under international GMP guidelines. Service quality is based on these practices and on the unique experience of the Association created ad hoc in Argentina to this end. PLACEMA was envisioned as sustainable long-term project whose services are fully accessible to the scientific community, the productive sector and to practicing clinicians.

Quality Control

PLACEMA genera sus productos celulares bajo estrictos controles de calidad. A continuación se describen dichos controles y se muestran resultados representativos. Este tipo de informe acompaña a los productos celulares que entrega PLACEMA:

  1. Karyotype
  2. Characterization of pluripotency I
  3. Characterization of pluripotency II
  4. Characterization of pluripotency III
  5. Characterization of pluripotency IV
  6. Characterization of pluripotency V
  7. Post-thaw viability.
  8. Mycoplasma
  9. Sterility testing for frozen iPSC clones

Test 1: Karyotype

Description

G-banding used to analyze parental and iPSC karyotype.


Specification for iPSC release

iPSC karyotype must match parental cell result.


Test 2: Characterization of pluripotency I

Description

iPSC pluripotency gene expression by RT-qPCR: SOX2 - OCT4 - HGDF3 - Rex 1 - pH34 - Nanog


Results

Pluripotency gene expression.
In figure:
iPSC Columns 1 to 4: independent iPSC colonies
PARENTAL Columns: seeding cells for reprogramming
No RT Column: internal negative control


Specification for iPSC release

Positive amplification for pluripotency-associated genes and for housekeeping gene, and negative amplification for parental samples and No-RT controls.

Negative result for parental genes. No-RT positive result for pluripotency genes or housekeeping gene (cDNA).

Test 2: Negative result for parental genes. No-RT positive result for pluripotency genes or housekeeping gene (cDNA).

Test 3: Characterization of pluripotency II

Description

Inmunocytochemistry of iPSC against pluripotency markers: Nanog - SSEA4 - OCT-4 - Tra 1-81


Results

iPSC inmunocytochemistry.
In figure:
A) OCT-4
B) Nanog
C) SSEA-4
D) Tra 1-81


Specification for iPSC release

Positive immunohistochemistry for pluripotency markers.

Test 3: Positive results for all markers tested.

Test 3: Positive results for all markers tested.

Test 4: Characterization of pluripotency III

Description

Generation of embryoid bodies by spontaneous differentiation of all three germ layers, in the absence of factors promoting pluripotency.


Results

The upper panel shows photographs of colonies obtained under a light microscope. Lower panel shows embryoid body formation at different days of the protocol.
Video of spontaneous generation of beating bodies.


Specification for iPSC release

Formation of embryoid bodies and positive germ cell staining in all three lineages.

Test 4: Embryoid body formation and specific lineage staining.

Test 4: Embryoid body formation and specific lineage staining.

Test 4: Video showing spontaneous formation of beating bodies.

Test 5: Characterization of pluripotency IV

Description

qRT-PCR of specific genes in the three germ cell layers of the embryoid body:
Endoderm: AFP y CXCR4
Mesoderm: Brachyury y PECAM1
Ectoderm: MAP2 y PAX6


Results

qRT- PCR to detect specific genes in each germ layer:
Table 1: Data on changes in gene expression between day 0 of differentiation and day 4.
Table 2: Statistical analysis of Table 1 results (one-tailed T-test).


Specification for iPSC release

Decreased OCT 4 expression (pluripotency marker), with increased expression of different differentiation markers for each germ layer.

Test 5: Negative Oct-4 results, with positive results for other markers.

Test 5: Negative Oct-4 results, with positive results for other markers.

Test 6: Characterization of pluripotency V

Description

Embryoid body inmunocytochemistry showing positive results for markers against all three germ lines under fluorescent microscope. Ectoderm: DCX y BetaIII Tubulina
Mesoderm: Desmina and Troponina
Endoderm: Gata 4 and AFP


Results

Inmunocytochemistry to detect specific markers for each germ line in embryoid bodies:
En figure
A) Dapi
B) DCX
C) BetaIII Tubulin
D) Dapi
E) AFP
F) Gata 4
G) DAPI
H) Troponin
I) Desmin


Specification for iPSC release

Shows positive staining for several differentiation markers of each germ line.

Test 6: Positive results for all markers tested.

Test 6: Positive results for all markers tested.

Test 7: Post-thaw viability.

Description

After thawing, number of cells showing uptake of vital stain and number of colonies formed in culture is counted.


Specification for iPSC release

Number of viable cells observed post-thaw must exceed 70%. Normal colony formation after thawing.


Test 8: Mycoplasma

Description

qPCR of genomic DNA extracted from human iPSC cells to detect mycoplasma contamination.


Results

Upper panel: PCR Housekeeping (expected product: 164pb)
Columns 1 to 7: reprogrammed DNA clones
Columns 8 to 12: control mycoplasma DNA (Sensitivity curve: 50, 5, 0.5, 0.05, 0.005ng)
Column 13: negative control
M: molecular weight marker 100pb
Lower panel: Mycoplasma PCRs (expected product: 370 a 490pb)
Columns 1 to 7: reprogrammed DNA clones
Columns 8 to 12: Mycoplasma DNA control (Sensitivity curve :50, 5, 0.5, 0.05, 0.005ng)
Column 13: negative control
M: molecular weight marker 100pb


Specification for iPSC release

Negative amplification for mycoplasma in DNA extracted from iPSCs, and positive amplification for control curve as well as for housekeeping gene.

Test 8: Negative assay for mycoplasma.

Test 8: Negative assay for mycoplasma.

Test 9: Sterility testing for frozen iPSC clones

Description

Frozen iPSC samples are placed in enriched culture media to induce growth and development of aerobic microorganisms. Viral controls are added under GMP guidelines.


Specification for iPSC release

Absence of microorganisms.