ISOLATES MESENCHYMAL STEM CELLS

GINA MARCELA CORDOBA

 The objective of the present study was to assess the feasibility of using
OBJECTIVE
adult DPCs for therapies to repair the corneal stroma.
 ISOLATES DPSC
 Dental pulp cells were isolated using the next procedure:
 Human adult third molars were collected after routine extraction at the University of Pittsburgh School of Dental
Medicine.
 The surrounding soft tissues were scraped from the teeth, the molars were cracked open, and the pulp was removed.
 The pulp tissue was minced and then digested for 1.5 hours at 37°C in a solution containing 1.6 × 10 3 U/ml type I
collagenase (Sigma) and 3.12 U/ml Dispase II (ZenBio).
 The digest was then passed through a cell strainer, and the dental pulp cells were plated at a density of 3,000 to 10,000
cells per cm2 and cultured in a growth medium containing high-glucose Dulbecco's modified Eagle's medium
(DMEM; Gibco) with 20% fetal bovine serum (Life Technologies, 10,000 U/ml penicillin, and 10 mg/ml streptomycin
(Gibco). The DPCs were passaged at 80% confluence using TrypLE Express enzyme solution (Life Technologies)
 Experiments were performed using cells at passages 2–5.
 Assess the effectiveness of different cell surface markers (CD51/CD140a, CD271,
OBJECTIVE STRO-1/CD146) in isolating DMSCs from DP and examined odontogenic and
chondrogenic potential of these isolated subsets of DMSCs.
 DPCs were cultured in alpha modified Eagle’s medium (a-MEM; Invitrogen, Carlsbad, CA, USA) containing
20% fetal bovineserum(FBS),non-essentialaminoacids, 100unitspermLpenicillin, and 100 units per mL
streptomycin, in a humidified 5% CO2 incubator at 37 6C (all reagents were from Invitrogen, Carlsbad, CA,
USA). Media was changed every 2–3 days, and cells were passaged at 80%–90% confluency

Fluorescence-activated cell Expression of stem cell surface markers in DPCs was determined
sorting by fluorescence-activated cell sorting (FACS) analysis.

The most effective marker was CD271, which isolated a

relatively large population of DMSCs (10.6%) and had the
strongest odontogenic and chondrogenic potential
It showed faster cell isolation:

 The pulp was digested with 2mg / ml

EDTA for 10 minutes, 4mg / ml type I
collagenase, 4mg / ml type II dispase for
40 minutes and 13ng / ml thermolysin
for 40 minutes. After the digestion of the
pulp, the culture was sonicated for one
minute.
1- Se seccionó la pulpa dental mecánicamente con una hoja de bisturí nº10.
2- Se recogieron los fragmentos y se realizó la digestión de estos con 2 mg/mL de
colagenasa tipo I durante 90 minutos (Método A) en condiciones de 37°C, 5% CO2 y
3% O2 (Figura 5.3).
3- Se centrifugó a 1000 g durante 2 minutos (Figura 5.4), y se recogió el precipitado
y cultivó en medio de cultivo completo a 37°C, 5% CO2 y 3% O2. La composición
del medio de cultivo es la siguiente: - DMEM con suplemento bajo de glucosa
(1mg/mL)............90% (v/v)
- Suero bovino fetal……………………..........……..……...10% (v/v)
En esta tesis se utilizó siempre el mismo medio de cultivo,
Dulbecco´s modified Eagle® (DMEM), con suplemento de
glucosa (1mg/mL) y suero bovino fetal.

Almacenamiento y transporte
Una vez extraída la pulpa dental, se almacenó en tubos de 15
mL estériles con medio de cultivo Dulbecco´s modified Eagle®
con suplemento bajo de glucosa (1mg/mL) y antibióticos
(Penicilina 100U/ml y Estreptomicina 100 µg/ml).

Cells were grown in a humidified 5% CO2 incubator at 37 °C

in Dulbecco’s modified alpha modified Eagle’s medium
(Invitrogen) supplemented with 15% fetal bovine serum (FBS;
Invitrogen).

Cell isolation and culture Primary dental pulp cells (DPCs)

were isolated from DP of extracted adult third molars (IRB#13-
000241-CR-00001) as previously described. DPCs were
cultured in alpha modified Eagle’s medium (a-MEM;
2- De todos los métodos empleados para su obtención, la
digestión de la pulpa con EDTA, colagenasa/dispasa,
termolisina y sonicación, fue el que demostró mayor velocidad
de aislamiento de las células.
3- Las células obtenidas de la pulpa dental fueron positivas para
los marcadores CD-133, Oct4, Nestin, Stro-1, demostrando sus
características de células madre mesenquimales. Además fueron
negativas para el marcador de células madre hematopoyéticas
CD-45. El marcador CD-34 fue positivo en un caso y negativo
en otro.
4- Las células madre de pulpa dental mostraron una tasa de
proliferación mayor al ser cultivadas bajo condiciones
fisiológicas de O2 (3%) que bajo condiciones ambientales (21%
O2).