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RAT BONE MARROW MESENCHYMAL STEM CELLS ISOLATION, 

CULTIVATION AND DIFFERENTIATION

Emoke PALL*, PhD Student

Professor Ioan GROZA*, PhD

Olga SORIŢĂU**, PhD, researcher

Ciprian TOMULEASA**, Student, researcher

Assistant Mihai CENARIU*, PhD

Daria GROZA***, PhD student

Teodora VLASIU*, PhD Student

Abstract

Bone marrow stromal cells (MSCs) represent a heterogeneous population derived from the non–blood-forming fraction of bone 
marrow that regulates hematopoietic cell development. In vitro, adult mesenchymal stem cells resident in this bone marrow fraction 
differentiate into bone, cartilage, and fat. Because MSCs can be easily obtained using a simple bone marrow aspiration and show 
extensive capacity for expansion in vitro, these cells have been considered as candidates for cell therapy. The aim of this study was 
to purify rat MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Our 
data demonstrate that we successfully isolated, culture-expanded and differentiated a relatively homogeneous population 

of MPCs from adult rat bone marrow.

Keywords: mesenchymal stem cells, culture expansion, osteogenic nodules, differentiation, rat bone marrow stem cells

Stem cell research has become an important field of study for molecular, cellular, and clinical 

biology as well as pharmaco-toxicology (1,3,4,5,8). This cells have a strong proliferative and unlimited 

self-renewal potential and are multipotent (4,8). 

Mesenchymal stem cells (MSCs) represent a population of the bone marrow microenvironment. 

MSC are non-haematopoietic stromal cells that were first isolated from the bone marrow (BM) but 

subsequently from other adult connective tissues (6,7) which have been explored as a promising 

* University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Department of Veterinary 
Reproduction, Obstetrics and Gynecology, 3-5 Manastur Street, 400372 Cluj-Napoca, Romania, tel. +40 264 596384 ext. 163, 
email: pallemoke@yahoo.com

** “Prof. dr. Ioan Chiricuţă“ Oncological Institute, Cluj-Napoca
*** “Iuliu Haţieganu“ University of Medicine and Pharmacy, Cluj-Napoca

Cluj Veterinary Journal, 15(1)/2009, pp. 29-32



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treatment in tissue regeneration. They exhibit multilineage differentiation capacity being capable 

to give rise to diverse cells like osteoblasts (2,9), chondrocytes, adipocytes, myocytes, tenocytes and 

possibly neural cells (7).

We describe a protocol for isolation of mesenchymal stem cells mononuclear cells from adult rat 

bone marrow.

Materials and Methods

MSCs were collected from the long bones of three adult Wistar rats (100- 150 g). The animals 

were killed by cervical dislocation. Their femurs and tibiae were carefully dissected of adherent soft 

tissue, the epiphyses were removed with a rongeur and bone marrow was flushed out by inserting a 

syringe needle (21G) into one end of the bone. Washing bone marrow were used DMEM/F12 (Gibco) 

medium supplemented with FCS 10% and antibiotic-antimicotic (100x) (Gibco)1%.

The aspirant was filtered through a 70 mm filter (Falcon) to remove bone fragments and then 

centrifuged at 1000 rpm for 10 min. The cell pellet obtained (containing both haematopoietic cells 

and marrow stromal cells) was then suspended in MSCs growth medium (DMEM/F12 1x, 10% fetal 

calf serum, 1% antibiotic-antimicotic). The cells were resuspended in normal culture medium to a 

final concentration of 5x106 viable cells per ml and were then plated on 75 cm2 flasks and left for 72 

h. The culture dishes were washed with PBS + 10% FCS to leave an adherent layer of cells containing 

marrow stromal cells. Confluent primary cultures were passaged, split, and re-plated; this cycle was 

repeated three times.

Osteogenic differentiation was induced by culturing MSCs for 2 weeks in DMEM (Gibco) 

supplimented with 10%FCS, 10-7dexamethasone, 10mM β-glicerophosphate, 1µg/ml insulin, 50µg/

ml ascorbic acid, 10ng/ml BMP2, 2ng/ml TGFβ, with a medium change every 2 days. At the end 

of the cultivation period, the cells were fixed with 4% paraformaldehide (Sigma) for 10 min. The 

cultures were examinated for identification of osteopontin positive cells and colonies according to 

the manufacturer´s instructions. They were then incubated separately with the primary antibodies 

to anti osteopontin (Sigma), followed by incubation with secondary fluorochromes FITC (anti-goat, 

diluted 1:100; Sigma). After antibody application, the cells were incubated with a Hoechst nuclear 

marker solution (diluted 1 mg/ml in distilled water; Sigma) for 15 min at 37°C. Following washing 

in PBS the preparations were mounted under coverslips, examined at high magnification under a 

fluorescence microscope (Zeiss Axioplan 2). 

Results and Discussion

For the mesenchymal stem cells recovery we used rats from Wistar line. The bone marrow was 

recovered by repeated washing of the medullar channel with DMEM/F12 supplemented with FCS. 

Rat MSCs obtained from standard purification methods and maintained in adherent culture consist 

of a morphologically heterogeneous population (Fig. 1A).

After 72 hours from the recovery was made the first medium change, after which was observed the 

emergence of two major subpopulations (Fig. 1 B,C)); cells with an elongated cells with two processes 

that extend in opposite directions from the cell body and pronounced bipolarity and other groups of 

polygonal cells with or without short processes. The first passage was made after 12 days (Fig. 1D) 

from the recovery when it was identified the emergence of some cellular colonies (Fig. 1C). 



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Fig. 1. Photomicrographs of rat mesenchymal stem cells after harvest 24 h (A), at 3 days (B), at 4 
days (C), small colonies (D), after 1st passage (E), 3nd passage (F)

After passage 3 (Fig. 1F) the culture was 

treated with osteogenic medium, and the medium 

change was made every other day (Fig. 2A). After 

4 days from adding the osteogenic medium we 

observed the emergence of some nodules (Fig. 2B) 

of different sizes irregularly dispersed.

After the examination with the inversed microscope was stated the growth in dimension of the 

nodules, fact also macroscopically visible. The cells around the nodules also suffered pronounced 

morphological modifications. Initially the cells presented a fusiform phenotype with some fine 

prolongations, followed by their transformation 

in round cells of different sizes. Towards the 

end of the experiment we observed a cellular 

juxtaposition: the fist layer of elongated cells 

over which it was stated the superposition of a 

layer of round cells. 

The osteogenic differentiation was identified 

with the aid of the osteopontin antibody. At 

the fluorescence microscope we identified the 

fact that the nodules formed were osteopontin 

positive that demonstrates that these nodules 

were formed from osteoblasts. 

Fig. 2. Phase contrast images of rat mesenchymal stem 
cells osteogenic differentiation; cells with morphology 
changed (A), and nodules of different sizes (B)

Fig. 3.- Immunohistochemical highlighting 
of osteopontin positive cells and nodules



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Concusion

In conclusions MSCs from rat bone marrow are relatively easily isolated from the bone marrow 

and can be manipulated in vitro, they could be good candidates for the development of various 

therapeutic modalities aimed to regenerate mesenchymal tissues. Better understanding of the molecular 

mechanism directing the differentiation of BMCs will eventually allow us to properly manipulate 

BMCs both in vivo and ex vivo for regeneration of complex tissues and organs. Less passaged BMCs 

cultured with osteoinductor medium proved to possess in vivo osteogenic potential. This can be 

applicable to use cell therapy in the repair of bone defects. In clinical feasibility, however, large cell 

numbers with osteogenic potential will be required. Further investigations are needed to establish 

the culture conditions that permit the rapid expansion of mesenchymal stem cells while retaining 

their potential for differentiation. 

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