الفهرس يوجد فقط 14 صفحة متاحة للعرض العام
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المستخلص
SUBJECTS AND METHODS
Study design:
It is an experimental study.
Site of study:
The study was carried out in the tissue culture unit in the Physiology Department, Faculty of Medicine, Suez Canal University after cord blood collection in the gynecology & obstetric department, Faculty of Medicine, and Suez Canal University.
Subjects :
Study population :
Normal volunteers.
Inclusion criteria :
1- full term pregnant women (col et al.,2003).
2- full term normal vaginal delivery (col et al.,2003).
Exclusion criteria:
1- Known history of hepatitis, infectious diseases, diabetes mellitus, severe hypertension, abortions or bad obstetric history (Col et al., 2003).
Sampling design
Sample size calculations:
The sample size was determined by using the following equation:
Sample size (n) = Z x 2
-------
(Dobson, 1984)
where:
: The standard deviation of CD34+ percentage given by 10 cm from the umbilical cord blood = 0.99 (Nimgaonkar M.T. et al., 1995).
Z: a percentile of standard normal distribution determined by 95% confidence level = 1.96
: The width of the confidence interval = 0.4
1.96 x0.99 2
Sample size (n) = -------------- = 24 samples.
0.4
Sampling method:
The study group is 24 umbilical cord blood samples.
Methods:
A) A good medical history was taken to be sure that the subjects are appropriate to be included in the study.
B) Cell sources:
Umbilical cord blood was obtained at the end of full term vaginal deliveries, after clamping and cutting of the cord (col et al., 2003)
The collections were made prior to the expulsion of the placenta. The UCB was collected while the placenta was still in utero. Using strict aseptic techniques the umbilical vein was cleansed with alcohol followed by betadine.
The umbilical vein was pierced and UCB collected into sterile collection tubes containing citrate phosphate dextrose adenine-l (CPDA-l) anticoagulant (approximately 2.5 ml) since total collection was approximately 10-20 ml.
* UCB units were stored at 4 °c & processed within 24 hrs. (col et al.,2003)
3-Separation and Purification of CD34+ HSC/HPC cells was carried out according To method described by ( Milteny et al., 1990 ) by Immunomagnetic separation technique : ( Dynal beads Oslo Norway ) :
* Principle :-
- Immunomagnetic cell selection using Dyna¬beads M-450 CD34 and DETACHaBEAD CD34 provides fast and reliable positive selection of (CD34+ hematopoietic progenitor cells. Dyna¬beads M-450 CD34 and DETACHaBEAD CD34 can be used to isolate CD34+ cells from a wide range of sample volumes.
- Upon mixing and incubation. (CD34+ cells bind to Dynabeads M-450 CD34 and the rosetted cells are isolated from the suspension with a DYNAL Magnetic Particle Concentrator (DYNAL MPC).
- A subsequent incubation with DETACHaBEAD CD34 gently detaches isolated cells from the beads.
- A DYNAL MPC is then used to separate the purified, positively selected CD34+ cells from the released Dynabeads M-450 CD34.
* Reagents used :-
- Isolation buffer: Phosphate buffered saline (PBS) without Ca2+ and Mg2+ with 2% bovine serum albumin (BSA), 0.6% tri-Na-citrate and 100 IU/ml penicillin streptomycin solution (PS).
- Culture Medium with 10% FCS, 100lU/mL PS: RPMI 1640 with 10% fetal calf serum (FCS), 100 IU/ ml PS
- Lymphocyte separation medium (ficoll hypaqe )
*Materials supplied:-
- Dynabeads M-450 CD34 are supplied as a suspension of 4 x 108 beads/ml in phosphate buffered saline (PBS), pH 7.4, containing 0.1% human serum albumin (HSA) and 0.02% sodium azide (NaN3).
- DETACHaBEAD CD34 are affinity, purified polyclonal antibodies against the FAB portion of mAb 561. It is supplied sterile filtered in PBS, pH 7.4. No preservatives are added.
**Additional material needed: Magnetic separation device [Magnetic Particle Concentrator (DYNAL MPC-I)].
**Dynal CD34 Progenitor Cell Selection System has the capacity to process 4 x 109 initial mononucleated cells ( MNC ) and to select up to 8 X 107 CD34+ cells per ml product of Dynabeads M-450 CD34.
FLOW CHART ISOLATION OF CD34+ CELLS
Isolate low density MNC
↓
Incubate with Dynabeads M-450 CD34
↓
Wash rosetted target cells.
↓
Release Dynabeads from target cells with
DETACHBEADS CD34
↓
Separate CD34+ cells from detached beads
↓
Collect and wash isolated CD34+ cells
**The procedure of separation and purifcation in details:
- The optimal concentration of Dynabeads re¬quired may vary for different. samples. The cell bead mixture should contain a concentration of 4 x 107 Dynabeads M-450 CD34/ml of MNC suspension.
A. Dynabeads washing procedure:
*The Dynabeads should be washed before use.
1. Resuspend the Dynabeads M-450 CD34 thoroughly in the vial.
2. Transfer the desired amount of Dynabeads into a tube.
3. Place the washing tube in a Dynal. MPC for 1 minute and pipette off the fluid.
4. Remove the tube from the Dynal MPC. Add 1-2 ml of washing buffer (PBS/0.1% BSA) and resuspend.
5. Repeat step 3 and resuspend the washed Dynabeads in an equal volume of washing buffer that was originally pipetted from the vial.
B. Preparation of cells:
1-. Collect CB in presence of anticoagulant.
*CB Samples should be diluted 1:4 in isolation buffer.
2. Transfer 7 ml of cell suspension on to 3 ml Ficoll and centrifuge 20 min at 800g.
3. Collect interface, suspend cells in an equal volume of isolation buffer and centrifuge20 min at 500g.
4. Resuspend pellet in isolation buffer again and centrifuge 20 min at 300g.
5. Resuspend pellet to a concentration in the range of 4 x 107 - 4 X 10 8 cells/ml in cold isolation buffer. The total volume should be at least 1 ml ,Maintain at 2-8°C.
C. Positive selection of CD34+cells ¬ Mixing of beads and cells:
1. Optimal concentration of Dynabeads M-450 CD34 is 4 x 107 bead / ml.
2. Resuspend cells thoroughly with a pipette with a narrow tip to prevent cells from aggregating before adding them to the beads.
3. Vortex cell- bead mixture 2-3 seconds to mix.
4. Incubate the cell bead suspension for 30 minutes at 2-8.C with gentle tilt rotation at10-20 rpm .
NOTE:
During incubation and separation it is important to keep the cell suspension and buffers cold (2-8°C) to prevent non¬specific attachment of phagocytic cells to Dynabeads.
Make sure the Dynabeads M-450 CD34 are kept in suspension during incubation.
D- Positive selection of CD34+cells Magnetic separation of rosetted cells:
1-After incubation, add isolation buffer to the height of the magnet.
2- Resuspend the cell- bead complexes. You may vortex for 2-3 seconds, if desired.
3- Place tube in a DYNAL MPC for 2 minutes to separate Dynabeads M-450 CD34-rosetted cells from non target cells.
4- Aspirate supernatant containing non-rosetted cells while the tube is still exposed to the magnet.
5. Repeat steps 1 - 4 above three times.
6- After the final wash, resuspend rosettes in 100 μl. of isolation buffer per 4 x 107 beads used , in a volume of at least 100 μl.
7- Proceed with detachment as described below:
b
Figure (VIII): a)Global View of MPC I. b) Tube exposed to the magnet
E. Detachment of beads from purified cells
1. Add 100 μI DETACHaBEAD CD34 per 4 x 107 Dynabeads M-450 CD34 in a volume of at least 100 μl. (i.e. use the same volume of DETACHaBEAD as used for resuspension of the rosettes).
2. Vortex 2-3 seconds to mix.
a) Incubate at room temperature with gentle tilt rotation for 45 minutes. After incubation proceed with step 3 below.
or
b) Incubate for 15 minutes at 37°C with gentle tilt rotation If rotation is not possible it is essential to shake the tube by hand every 5 minutes to keep rosettes in suspension. After incubation proceed with step 3 below.
NOTE:
Detachment will become less efficient if the incubation temperature is below20°C.
Due to relatively small sample volume care should be taken that the cells remain near the bottom of the tube to avoid loss of cells.
3- After incubation, add 2 ml of isolation buffer and vortex 2-3 seconds to enhance detachment of Dynabeads M-450 CD34 from the cells.
4. Place the tube on the DYNAL MPC and, allow the Dynabeads M-450 CD34 to accumulate on the tube wall for 2 minutes.
5. Transfer released cells to a new tube Repeat steps 3-4 three times, pooling released cells into a single tube.
6. Place the tube with the pooled isolated cells on the DYNAL MPC for 2 minutes to remove any residual beads.
7. Transfer supernatant containing released cells to a fresh conical tube.
F. Washing positively selected CD34+ cells:
1- Wash the cells twice in 10 ml or more of isolation buffer, centrifuging 10 min at400g to pellet cells
2- To avoid cell loss, leave 100 μI fluid over the pellet when removing the supernatant. Avoid decanting the supernatant as it may lead to loss of cells.
3. After final centrifugation, resuspend pellet in l ml isolation buffer.
4-Assessment of the quantity & the quality of the separated cells:
Quantity by: putting the sample on a cell counter then its staining by using Geimsa stain (Frans et al., 2005).
Quality by: assessment of the viability using tyrpan blue dye exclusion test (Donna et.al., 2005).
5-cryopreservation:-
** UCB MNC were cryopreserved using the following procedures:-
- 0.8 ml from separated cord blood MNC was cryopreserved By using 6% hydroxy-ethyl starch (HES) & 5% DMSO ( Nademanee et al.,1991) according to the following detailed procedure:
a) 0.8 ml. CB was added slowly to 1.2 ml of freezing medium. This freezing medium was prepared immediately before use (167 micro liters of pure DMSO and 2 ml 10% H.ES).
b) The mixture of the CB in freezing medium was then directly added into a cryovial ampoule and stored for 1 hour in (¬-80°C) deep freezer in As immediate freezing is important to prevent apoptosis which develops relatively quickly after isolation from cord blood0 (Paczkowska E.,2002).
c) After one hour, the ampoule was kept in the vapor phase of the liquid nitrogen, to allow further freezing until analysis (Ineke Slaper Cortenbach, Ph.D. Personal communication).
d) One month later, thawing of mixture was done according to the following detailed procedure:
e) The bone marrow mixture cryovials were thawed by immersion in a 37°C water bath and transferred into a larger tube.
f) 1.5 ml phosphate buffered saline (PBS without Ca++ or Mg++) was added DROP wise slowly while holding tube and gently swirling.
g) Tube was filled to 5 ml with PBS. Gently tube was inverted to mix.
h) Cells were spined down at 1,200 rpm for six minutes.
i) Supernatant was discarded and tube was flicked gently to re suspend the pellet.
j) After thawing, the CB samples were subjected for the following procedures:
** Separation of CD34 hematopoietic stem cells by Immunomagnetic beads as described above in details.
6-Assessment of the quantity & the quality of the separated cells after thawing by using the same procedures to determine the post cryopreservation results .
DATA ANALYSIS
. A computer aided statistical analysis of results was done using the statistical programs (Excel, SigmaXL and SPSS).
. Data were presented as means, standard deviations (SD), minimum and maximum for the numerical analysis.
. Correlation between two variables was done using correlation coefficient test.
. Comparison between two groups was done using student’s t-test.
. The level of statistical significance was: P < 0.05 significant.
ETHICAL CONSIDERATIONS
*Informed consent and confidentiality:
- The purpose of the study will be explained and then a consent will be taken from every participant in the study.
-The results will be confidential and no personal data will be published.
*The investigator will give the medical authorities of the hospital a copy of the results.
*The measures will be used are safe and with no harm to the participants.
SUMMARY
* In spite of many studies were done world wide in order to establish cord blood banks, No Published Egyptian studies were done in the field of stem cell. So, this study was carried out to establish a tissue culture unit in the Physiology department, Suez Canal University, Faculty of Medicine to study the effect of cryopreservation on CD34+ cell yield & viability.
* Twenty-four UCB samples were taken from female volunteers attending the obstetric & gynecology department for full term vaginal deliveries, samples were assessed for CD34 + cells count & viability before & after cryopreservation.
*Before cryopreservation, CD 34 + cell yield was 3 +1.71 x 105/ml with a range from 1x 105/ml to 6 x 105/ml & viability% was 87.08% + 4.75% ranging from 80% - 90%
*After cryopreservation, CD34 + cell count was 2.08 x 105 + 1.64/ml ranging from 0 - 5x105/ml & viability % was 78.33% + 5.25% ranging from 70% - 90%
This gave a conclusion, that cryopreservation affects the CD34+ cell count & viability.
It was found that male sex adversely affects CD34 + cell count but weight has no correlation with CD34+ cell count.
So, other researches are needed to study how to optimize the conditions affecting CB collection & storage.
الملخص العربي
في منتصف الثمانينيات ، تم اكتشاف المستضد الخلوي الموجب+ 34 ،الذي يوجد علي سطح الخلايا الدموية الغير ناضجة بما تشمله من الخلايا الجذعية الدموية القادرة علي التحول إلي نوع واحد أو عدة أنواع أنواع من الخلايا.
في السنوات الأخيرة ، ظهر الجبل السري كبديل حيوي للخلايا الموجبة للمستضد الخلوي 34 في حالات زرع النخاع خصوصاً في المرضي الذي ليس لديهم توافق مع المتبرعين بنخاع العظم.
وعلي النقيض من إجراء العديد من البحوث في مختلف بقاع العالم من أجل إنشاء بنوك للدم، فإنه لا يوجد أي معلومات منشورة في هذا المجال في مصر، كذلك قمنا بهذه الدراسة في وحدة زراعة الأنسجة ، قسم الفسيولوجي، كلية الطب ، جامعة قناة السويس من أجل دراسة تأثير الحفظ علي عدد الخلايا وحيويتها.
في هذه الدراسة، 24 عينة تم أخذها من الحبل السري للأطفال الذين تمت ولادتهم من السيدات أصحاب تاريخ صحي خالي من الأمراض وأصحاب ولادات طبيعية لأطفال تامين النمو.
- تم تقييم الخلايا من حيث العدد والحيوية قبل وبعد الحفظ وكانت النتائج كالأتي:
- قبل الحفظ في العينات الطازجة :
- متوسط عدد الخلايا الموجبة للمستضد الخلوي + 34 كان 3× 10 5 + 1.71 /مل بمعدل يتراوح ما بين 1×10 5 / مل – 6 × 10 5 / مل
- حيوية الخلايا كانت بمتوسط 87.08% +4.75 % بمعدل يتراوح ما بين 80% - 90%
- بعد الحفظ في العينات المحفوظة :
- متوسط عدد الخلايا الموجبة للمستضد الخلوي + 34 كان 2.08 × 10 5 + 1.64 /مل بمعدل يتراوح ما بين صفر – 5× 10 5 /مل
- حيوية الخلايا كانت بمتوسط 78.33 % + 5.25% بمعدل يتراوح ما بين 70% - 90%
- من السابق ، نجد أن حفظ الخلايا يؤثر علي عددها وعلي حيويتها.
- وجد في هذه الدراسة أن الأولاد الذكور لهم تأثير علي زيادة عدد الخلايا ولكن الوزن ليس له علاقة بعدد الخلايا الموجبة للمستضد الخلوي + 34
- وبالتالي نحتاج العديد من الدراسات الأخرى من أجل تطوير الظروف المؤثرة علي تجميع وتخزين دم الحبل السري.