An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Summary

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Abstract

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug – clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Introduction

Mesenchymal stem cells (MSCs) play a major role in regenerative medicine and tissue engineering. They can migrate, differentiate into various cell types ¹ and engraft, which renders them the ideal candidates for autologous therapies ^2,3. Lately, clinical trials using MSCs for bone and cartilage repair, graft versus host disease or heart disease were launched ⁴. These MSCs can be harvested from the umbilical cord or adipose tissue but most promising results were obtained from bone marrow derived stem cells ⁵.

The iliac crest allows to collect a considerable amount of bone marrow and therefore serves as main site of aspiration ⁶. However, the quality of the aspirate decreases with increasing volume of bone marrow withdrawn. While the first 5 ml of bone marrow aspirate contain MSCs of high quality, withdrawal of larger volumes leads to dilution of the aspirate with peripheral blood from the highly vascularized bone ⁷. Because of the present megakaryocytes and platelets, bone marrow aspirates are prone to clotting, unless anticoagulants are used. But even with anticoagulants, clots may occur.

In bone marrow, MSCs represent only a small proportion of the total cell pool ⁸ and have to be expanded in culture for most tissue engineering or therapeutic applications ⁴. The quality of such a culture largely depends on the initial cell pool, i.e., diversity and a high starting number ⁹. Low numbers of MSCs from withdrawals may be partly explained by donor variability. On the other hand, MSCs from low quality samples require longer time in culture and extended passaging to reach the desired number of cells. In either case, extended passaging is a source of cell senescence and can lead to the loss of differentiation potential ¹⁰. Therefore, optimized protocols that can maximize cell yield and prevent from detrimental effects have to be developed ^11,12.

When we began to work with canine MSCs, we were astonished to see that about three in four canine bone marrow samples contained clots, while fortunately clotted human samples (one in ten) were less frequent. On the other hand it was no surprise, that we observed much lower yields of MSCs from clotted samples. To solve the recurring issue of clotted samples, we developed the protocol using the thrombolytic drug urokinase instead of resampling.

Thrombolytic therapies can counteract life threatening situations such as occlusion of blood vessels causing heart attack, stroke or embolisms because of unwanted clotting. They work by degradation of the clots through enzymatic cleavage of fibrin by plasmin and enzymatic plasminogen activators. Despite the wide use for treatment of patients, only very few publications exist that utilized thrombolytic activities for laboratory applications to rescue clotted samples, mostly focusing on lymphocytes. In 1987, Niku et al. described the use of streptokinase for dissolving blood clots resulting in functional lymphocytes ¹³ and four years later, De Vis et al. extended the use of streptokinase to isolate leukemia cells from blood and bone marrow for flow cytometric applications ¹⁴. A more recent publication suggests the use of Alteplase for cancer diagnostics ¹⁵. While using the same enzymatic approach, our protocol focuses on the isolation of multipotent MSCs form bone marrow to provide a tool for researchers in the stem cell field.

Protocol

NOTE: Human bone marrow aspirates from the iliac crest were collected from consenting donors with the approval of the ethics committee of the canton of Lucerne. Canine bone marrow aspirates from the iliac crest were collected with dog owner’s consent.Human (approx. 20 ml) and canine (approx. 10 ml) bone marrow aspirates were anti-coagulated by addition of 15 ml of 3.8% sodium citrate immediately after withdrawal in the operation theatre. The samples were transferred to the laboratory environment for processing the same day as withdrawn.

1. Preparation of Urokinase (Prior to 1^st Use)

1. Reconstitute urokinase using sterile phosphate buffered saline (PBS) according to the manufacturer’s instructions: Add 10 ml PBS to the septum-inlet flask using a 10 ml syringe. Dissolve the dry substance by swirling to obtain 50,000 injection units (U) per ml.
2. Prepare 500 µl aliquots (25,000 U each) into sterile tubes. Store aliquots -20 °C and use it when required for at least 6 months.

2. Preparative Steps (Prior to Every Bone Marrow Treatment)

1. Thaw one aliquot of urokinase (25,000 U) per clotted bone marrow aspirate (aspirate volumes up to 25 ml have been successfully treated using a single aliquot of 25,000 U).
2. Preheat a water bath or shaking incubator to 37 °C.

3. Enzymatic Digest of the Clot

1. Perform all work in a laminar flow biosafety cabinet to avoid microbial contamination of the bone marrow samples during processing. When working with potentially infectious human material, the use of protective gloves as well as careful handling is advised.
2. Place a 100 µm cell strainer on top of a sterile 50 ml tube. Carefully pour the bone marrow aspirate through the cell strainer, tilting the strainer or moving around the clot material. Use a sterile pipet tip for better flow through the filter mesh.
   NOTE: Avoid any dilution of the clot, e.g., by washing the clot with PBS. Urokinase acts indirectly and requires components of the serum (i.e., plasminogen) from the biopsy for effective digest. While continuing the procedure with the clot material, the filtrate can be kept at RT until further use in step 3.6.
3. Transfer the bone marrow clot material into an empty cell culture dish using sterile forceps. Cut the debris into small pieces of approximately 2 mm³ using a sterile scalpel.
4. Transfer the small pieces of the clot into a 50 ml reaction tube using the sterile forceps. Ensure that the minced clot has a wet appearance. If it appears to be dry, use some of the filtrate from step 3.1 to moisten.
5. Triturate the clot by pipetting up and down for 5 times using a 5 ml microtiter pipet. Add 1 aliquot of urokinase to the sample. Incubate for 30 min at 37 °C either in a water bath or in a shaking (gently) incubator.
6. Triturate by pipetting up and down for 5 times using a 5 ml pipet. Perform this step in a biosafety cabinet. Incubate for another 30 min at 37 °C. After the incubation, triturate again 5 times using a 5 ml pipet. Pass over a fresh 100 µm cell strainer to pool with the filtrate from step 3.2.
7. Centrifuge the cell suspension at ambient temperature for 10 min at 500 x g. Discard the supernatant. Re-suspend the cell pellet in media as described below or according to the standard procedure of your laboratory for MSC expansion culture.

4. Seeding for Expansion Cell Culture and CFU Plates

1. Resuspend the cells (consisting of erythrocytes and mononuclear cells) in 50 ml of basal medium: Alpha-MEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml amphotericin B. Count the cells diluted 1:10 in Trypan Blue solution using a Neubauer chamber.
2. Plate the cells in cell culture flasks at the density of 5 x 10⁷ cells/cm² and incubate in a humidified incubator at 37 °C. If available, use hypoxic (5% oxygen) conditions. For the CFU assay control, plate 10⁹ cells in a cell culture dish of 10 cm diameter.
3. After 3 days of culture, mesenchymal stem cells are attached to the cell culture dish while other cells remain in suspension. Change the media with basal medium supplemented with 5 ng/ml basic fibroblast growth factor (bFGF). For the CFU assay control, treat the cell culture dish likewise.
4. Continue cell culture for a total of 2 weeks, exchanging media three times a week. If cells exceed 80% confluency, split by trypsinization. For the CFU assay control, exchange the media likewise, but do not split the cells for the course of the two weeks.

5. Giemsa Stain for CFU Assay

1. Prepare 10 ml of Giemsa-solution per dish: dilute the stock solution (7.6 g/l Giemsa in glycerol:methanol) 1:10 in sterile water (always prepare a fresh working dilution).
2. Remove the media from the cell culture dish and wash the cells with PBS. Be very precautious when applying liquid to the petri dish and avoid washing off the cells.
3. Fix cells in pure Methanol for 5 min at RT. Discard the Methanol. Add the Giemsa-solution and incubate for 60 min in a humidified incubator at 37 °C.
4. Wash two times with PBS. Air dry the plate head first on a paper towel. Count the colonies manually. This is best achieved using a marker pen on the back of the plate.

6. MSC Differentiation

1. Canine MSCs differentiation
   NOTE: Canine MSCs were differentiated into chondrogenic, osteogenic and adipogenic lineages by stimulation with the appropriate media for four weeks.
2. For Adipogenic differentiation, culture MSCs in monolayer at 4 x 10⁵ cells/cm² alternating two different culture conditions as follows;
   1. Culture in adipogenesis maintenance media containing DMEM + GlutaMAX, 3% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, 2.5 µg/ml amphotericin B and 170 mM insulin.
   2. Culture in adipogenesis inducing media with maintenance medium supplemented with 3% FBS, 5% rabbit serum, 1 µM dexamethasone, 500 µM 3-Isobutyl-1-methylxanthine, 33 µM biotin, 5 µM rosiglitazone and 17 µM pantothenate ¹⁶.
   3. Revel the lipid droplets by staining with Oil Red-O. Briefly, fix cells with 10% formaldehyde, wash with PBS and stain with 0.35% Oil red O in isopropanol.
3. For Osteogenic differentiation culture MSCs in monolayer at 7 x 10³ cells/cm² and stimulate in the following:
   1. Advanced DMEM (GIBCO) + GlutaMAX, 5 % FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, 2.5 µg/ml amphotericin B, 50 µM L-ascorbic acid 2-phosphate, 10 mM ß-glycerophosphate and 100 nM dexamethasone.
   2. Identify the mineralization deposits by Von Kossa stain (5 % AgNO₃). Briefly, fix the cells with 10% formaldehyde, wash with PBS and stain with 5 % AgNO₃in distilled water.
4. Chondrogenic differentiation
   1. Cut cubes (3 mm per side) from a sponge shaped medical device, constituted from lyophilized collagen type I, and used as scaffold material to support cells ¹⁷.
   2. Seed MSCs on the top of the cubes at the concentration of 4 x 10⁶ cells/ml (~70,000 cells/cube).
   3. Prior to the addition of media, allow the cells to adhere to the cubes for 30 min.
   4. Maintain MSC-collagen constructs in chondrogenic media consisting of DMEM/F12 + GlutaMAX, 2.5 % FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, 2.5 µg/ml amphotericin B, 40 ng/ml dexamethasone, 50 µg/ml ascorbic acid 2-phosphate, 50 µg/ml L-proline, 1x Insulin-Transferrin-Selenium X, and 10 ng/ml transforming growth factor-β1.
   5. Use alcian blue staining to visualize accumulation of proteoglycans in constructs sections. Briefly, stain the sections O/N with 0.4% alcian blue dissolved in 0.01% H₂SO₄ and 0.5M guanidine hydrochloride. Next, wash the sections were washed for 30 min in 40% DMSO and 0.05M MgCl₂. Cells were counterstained with nuclear fast red.

Results

The facts that 74% of canine bone marrow samples (n=54) contained clots when they arrived in our laboratory (Figure 1A) along with decreased MSC yields from these samples, made us believe that a considerable number of MSCs was trapped within the clots. Indeed, a simple DAPI-stain of sectioned clot material confirms the presence of nucleated cells in high density (Figure 1B). This ultimately leads to low numbers of MSCs available for expansion culture, which triggered us to develop the pr...

Discussion

Routinely we sample bone marrow while the patient is undergoing surgery (in our case mainly spine surgery), with the advantage that only little additional work has to be carried out by the personnel in the operation theatre. Even though the samples are mixed with sodium citrate immediately after withdrawal, many samples were partially clotted when they arrived in the laboratory for processing. At this stage, resampling to replace clotted specimens would be a separate additional intervention necessitating again local or g...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Basal Medium Components
PenStrep 100X	Gibco	15140122
Human FGF-basic	Peprotech	100-18B
MEM Alpha w/ Nucleoside, w/ stable Glutamine	Amimed	1-23S50-I
FBS Heat Inactivated	Amimed	2-01F36-I
Amphotericin B	Applichem	A1907
Adipogenic Medium Components
DMEM-HAM F12 + GlutaMAX	Amimed	1-26F09-I
Insulin	Sigma	I5500
Rabbit serum	Gibco	16120099
Dexamethasone	Applichem	D4902
3-Isobutyl-1-methylxanthine	Sigma	I5879
Biotin	Sigma	B4639
Rosiglitazone	Sigma	R2408
Pantothenate	Sigma	P5155
Oil Red-O	Sigma	O0625
Osteogenic Medium Components
L-ascorbic acid 2-phosphate	Sigma	A8960
ß-glycerophosphate	Sigma	G9422
Silver nitrate (AgNO3)	Sigma	S6506
Chondrogenic Medium Components
Biopad - sponge shaped medical device	Euroresearch
L-proline	Sigma	P5607
Insulin-Transferrin-Selenium X	Gibco	51500056
Human transforming growth factor-β1	Peprotech	100-21
Alcian Blue 8GX	Sigma	A3157
Nuclear fast red	Sigma	N8002
Generic
Tri-Sodium citrate dihydrate	Applichem	A3901
PBS	Applichem	964.9100
Urokinase	Medac	1976826
0.5% Trypsin-EDTA	Gibco	15400054
Giemsa stain	Applichem	A0885
Formaldehyde	Applichem	A0877
Sulfuric acid (H2SO4)	Applichem	A0655
Dimethyl sulfoxide (DMSO)	Applichem	A1584
Magnesium chloride (MgCl2)	Applichem	A3618
Guanidine hydrochloride	Applichem	A1499
Consumables
50 ml reaction tube	Axygen	SCT-50ML-25-S
10 ml syringe	Braun	4606108V
Sterican needle (22G)	Braun	4657624
1.7 ml Microtubes	Brunschwig	MCT-175-C
100 μm cell strainer	Falcon	6.05935
sterile forceps	Bastos Viegas, SA	489-001
sterile scalpel	Braun	5518059
Primaria cell cuture dish	Falcon	353803
C-Chip Neubauer Improved	Bioswisstech	505050
cell culture flask - Flask T300	TPP	90301
Equipment
Microbiological biosafety cabinet class II	Skan	82011500
water bath	Memmert	1305.0377
Stripettes Serological Pipette 5ml	Corning	4487-200ea
microscope	Olympus	CKX41
humidified incubator Heracells 240	Thermo scientific	51026331
Heraeus Multifuge 1S-R	Thermo scientific	75004331